The mean transpiration rate (E) varied significantly (0.05) among the cultivars and the phenological stages at both locations; however, it was not affected by yearly variations, as the responses in both vintages varied non-significantly (Figure 8 and Table 2). A higher transpiration rate (E) was recorded

Table

Leaf chlorophyll content is usually stable; however, it is affected by heat stress, as is leaf growth and other physiological attributes, after a threshold temperature is attained. In comparison to 2019, lower temperatures, higher rainfall and more humid conditions during the 2020 vintage favoured chlorophyll production. These results are in line with a study by Carvalho et al. (2015), in which a stepwise heat stress and recovery experimental setup for two wine grape varieties, Tourina Nacional and Trincaderia, resulted in heat stress at 42 °C reducing leaf chlorophyll contents; interestingly, the grapevine leaves tended to recover when exposed to favourable conditions at 25 °C and chlorophyll levels increased in a stepwise accumulation and recovery process. Significantly lower CCI was recorded in the colder location of Islamabad than in Chakwal. This difference was small, but it indicates that, in addition to seasonal variability for temperature, other factors, such as soil type, water and nutrient availability, also contributed to variation in leaf chlorophyll levels (Gruber and Kosegarten, 2002; Sánchez et al., 2020). For instance, soils with higher clay content and lower pH have sub-optimal Fe content, which may negatively affect leaf chlorophyll levels, as was observed in the vineyards of the Mediterranean region (Casanova-Gascón et al., 2018). Similarly, in our case, the soil at Islamabad has a higher clay content than that of Chakwal. During the physiological monitoring of the vines, more vegetative growth was observed in the grapevine cultivars at Islamabad than in those at Chakwal; this might be due to the less favourable climatic conditions in Chakwal, where T max reached up to 40 °C during the summer, negatively affecting vine growth. Given that the same pruning technique was applied to each vine so that 32 to 40 buds per vine remained, further research to evaluate the relationship between leaf chlorophyll and vine vigour, pruning type and bud charge is needed to better understand this behaviour. The lower leaf chlorophyll content observed in Islamabad compared to Chakwal is not likely to have affected the photosynthetic potential of the leaves; Walker et al. (2018) found that grapevine canopies can assimilate similar amounts of carbon dioxide with significantly lower leaf chlorophyll levels and can also save up to 9 % of leaf nitrogen. In future studies, it would be useful to explore the relationship between vine vigour and leaf chlorophyll levels and soil nitrogen (N) availability, focusing on the repartitioning of N from chlorophyll towards more beneficial investments in order to accelerate the rate of carboxylation and electron transport reaction. Moreover, different cultivars growing under similar climatic conditions showed varying chlorophyll levels; for instance, cv. NARC Black had the highest leaf chlorophyll content. Such genotypic variability for leaf chlorophyll has been highlighted by Carvalho et al. (2015) and Filimon et al. (2016): wine grape var. Tourina Nacional contained significantly more chlorophyll than did var. Trincaderia. Moreover, in the present study, CCI also varied depending on phenological stage - from bud burst to maturity - with the highest mean values at harvest. Similar increases in chlorophyll content from bud burst to veraison was observed by Filimon et al. (2016) for table grapes var. Gelu, Milcov, Cetatuia and Napoca. However, in our case, there was a slight increase in chlorophyll content at maturity which might be due to more rainfall near ripening promoting chlorophyll production, even near maturity. In a related study, Chavarria et al. (2012) also observed higher chlorophyll content near maturity.

Higher internal leaf CO₂ or sub-stomatal conductance (Ci), particularly for the warmer location (Chakwal) and the hotter vintage (2019), indicate that leaf Ci activity is positively related to temperature. These elevated Ci levels under high temperature conditions are in line with the work of Luo et al. (2011), in which higher temperatures (35-45 °C) were found to increase leaf Ci activity. Similarly, Greer (2018) observed that internal CO₂ activity (Ci) was highest at a lower temperature (20 °C), but it decreased at 30 °C and then stabilised at 35 °C. Ci has also been shown to increase at temperatures above 40 °C (Greer, 2012; Greer and Weedon, 2012). The elevated Ci levels at high temperature have a negative relationship with leaf photosynthetic activity (A); this trend is in line with Greer and Weedon (2012b), who also observed a negative relationship between Ci and A under increasingly hot climatic conditions. In future research on these cultivars, it would be worth focusing on quantifying the Ci and A relationship under the influence of elevated air temperature conditions. In the present study, the internal Ci concentration of the grapevine cultivars also varied, with relatively higher levels being recorded for cvs. NARC Black and King's Ruby compared to Perlette and Sugraone. Similar genotypic variability in grapevine cultivars in different temperature regimes has been highlighted by Greer (2018). It is interesting to observe that the cultivars Kings Ruby and NARC Black, which had significantly higher Ci values, showed visual signs of heat stress, while Perlette and Sugraone, which had lower Ci values, did not show any visual signs of summer heat stress until harvest. In terms of phenological stages, Ci was at its highest at the early developmental growth stages (i.e., 298 µmol/m²/s^{at the5-leaf stage), and near maturity (287 µmol/m2/s); however, the values were significantly lower at the other stages particularly at full bloom and berry set. These results partially correlate with the work of Greer (2012), in which Ci varied during the season with vine growth, being at its highest at the beginning of the season, then decreasing during the season; however, it increased again near maturity.}

Leaf stomatal conductance showed high variability between the two locations in the 2019 vintage, which can be linked to their differing temperatures, particularly during the hot summer; meanwhile, there was less leaf stomatal conductance variability between locations in the 2020 vintage. This might be due to the smaller number of days of heat stress in the 2020 vintage, particularly at the warmer location (Chakwal). These findings are in line with those of Luo et al. (2011), who observed a decline in stomatal conductance for temperatures above 35 °C. In the present study, the stomatal conductance of cv. Sugraone was exceptionally high at Islamabad in 2019, due to higher T max (39 °C) on 5 May (berry set) and increased daily evaporation (6.5 mm), compared to the other cases (locations and years) at the onset of the berry set stage, when T max was low (28.5 °C to 33 °C) with low evaporation (2.0 mm to 5.6 mm). These findings are in line with those of Greer (2012) and Greer and Weedon (2013), who concluded that high temperature above 35 °C may likely increase leaf stomatal conductance. In a related study by Sabir and Yazar (2015), stomatal conductance of grapevine cultivars was found to increase from early morning (08:30) to mid-morning (10:30), when a peak value was attained, which then gradually decreased; the stomatal conductance ranged from 96.7 to 287.7 mmol/m²/s. For Vitis labrusca var. Niagara and Rosada grown in Brazil, stomatal conductance increased from 8:00 (morning) to 12:00 (noon), but was negative during the afternoon (Deus et al., 2016). Leaf stomatal conductance also varies during the growing season, as reported in this study and discussed by Greer (2018). In another study, Greer (2012) found that stomatal conductance increased from 20 to 25 °C, and then stabilised before decreasing at 35 °C. However, a further rise in temperature slightly increased stomatal conductance. The stomatal conductance varied significantly between cultivars; similar genotypic variability for leaf stomatal conductance was observed by Sabir and Yazar (2015) and Greer (2018). Leaf stomatal conductance also varied depending on key phenological stage, with significantly high values at berry set and low values at harvest, which may correspond to the prevailing temperature and plant demand for gaseous exchange at different developmental stages.

Net photosynthetic activity was significantly higher at the relatively cold location of Islamabad than at Chakwal during the colder vintage of 2020. The lower photosynthetic activity for the hotter vintage and colder location indicates the negative effects of high temperature on grapevine physiological processes. These findings are in line with those of Luo et al. (2011), who observed that high temperatures above 35 °C decreased photosynthesis and that at a temperature of 45 °C the acceptor site of PSII was inhibited. Greer (2012) found net photosynthesis to increase with rising temperature from 15 to 35 °C, with an optimum range of 25 to 30 °C, and to sharply decrease by 30 % (cv. Shiraz) and 60 % (cv. Semilon) at 45 °C. Net assimilation in grapevine has been found to peak during the summer at an optimum photosynthesis temperature of 30-35 °C (Greer, 2019; Greer, 2020). Moreover, periods of heat stress can also affect vine photosynthetic responses; for example, heat stress at 25/40 °C for 4 days caused a 35 % decline in photosynthesis, with a subsequent 12-day recovery period (Greer and Weston, 2010). Photosynthetic activity varied significantly between cultivars: cvs. NARC Black and Sugraone showed higher photosynthesis values than the other cultivars. Similar genotypic variability for photosynthetic activity has been observed in four table grape cultivars, Crimson seedless, Superior seedless, Razaki and Horoz Karasi (Kamiloğlu et al., 2014), in wine grapes cvs. Chardonnay and Merlot (Greer, 2018) and in Muscadinia rotundifolia var Carlos and Noble in Florida (Colova et al., 2007).

Photosynthetic activity varied depending on key phenological stage, being significantly high at berry set and near blooming stage, but lower at harvest, which may correspond to variable temperature above critical thresholds. Grapevine developmental stages are known to vary in terms of optimum temperature ranges; for example, 25-30 °C is the optimum range for early growth after bud burst, but 30-35 °C is the optimum window for most phenological stages (Greer, 2020). Heat stress negatively affects photosynthetic activity in key phenological stages as observed by Greer and Weston (2010): vine photosynthetic activity decreased from 11 µmol/m²/s to 2 µmol/m²/s when nearing the maturity stage.

High transpiration is an indicator of an effective photosynthetic process and the plant's ability to perform its physiological processes efficiently. The observed data indicate that the vines at the warmer location (i.e., Chakwal) were subject to conditions of stress (elevated temperature and lower rainfall), which might have caused partial stomatal closure, thus low transpiration. In a related study on Vitis labrusca var. Niagara Rosada grown in Brazil, an increase in temperature by 2.3 °C significantly increased leaf transpiration at 08:00 (morning), but at 12:00 (noon) negative values for transpiration were obtained due to the higher temperatures (Deus et al., 2016). While elevated temperatures increase leaf transpiration (Greer, 2012; Greer and Weedon, 2013), they usually act in conjugation with water stress owing to low seasonal rainfall and can hence potentially reduce the transpiration potential of plants in warmer conditions (Kamiloğlu et al., 2014). This is because the transpiration rate of grapevine cultivars vary significantly, as was observed in present study. Similarly, Greer (2018) observed 50 % higher transpiration in var. Merlot leaves than in those of var. Chardonny.

Leaf transpiration rates also varied depending on key phonological stage, being significantly at their highest at berry set and the full bloom stages, possibly due to higher plant demand for gaseous exchange. Moreover, they corresponded to higher temperatures, partial stomatal closure, and relatively low gaseous exchange. Significant variations in leaf transpiration at different growth stages were observed by Andrianasolo et al. (2016) in sunflowers. However, the present research findings do not agree with those of Thapa et al. (2017), who observed non-significant differences in the transpiration efficiency of sorghum at the six-leaf, flag-leaf, grain-filling and maturity stages. The high sensitivity of the transpiration activity of grapevine compared to other crops could be one reason for these varied responses.

WUE is a critical physiological attribute of vines, reflecting the ratio of carbon assimilated during photosynthesis to transpiration losses for water (Flexas et al., 2010). In the present study, WUE was significantly higher at the colder and more humid location of Islamabad than at Chakwal, and it was higher during the 2020 vintage, which had lower temperatures than in the 2019 vintage. These findings are in line with those of Medrano et al. (2015), who observed that high temperatures and water stress negatively affect leaf WUE. The vines managed through the anti-transpirant strategy of applying Kaolin have up to 18 % more WUE, which reduces the adverse effects of heat and water stress, such as sun burn and berry shrivel (Brillante et al., 2016). Genotypic variability among grapevine cultivars affects vine WUE, as was observed in this study, with WUE being significantly higher in cvs. NARC Black and Sugraone than in cv. Kings Ruby. These findings are in line with those of Kamiloğlu et al. (2014), who also observed WUE to vary in different table grape cultivars. The inherit genetic variability of 23 grapevine genotypes in terms of WUE and stomatal conductance under Mediterranean climatic conditions of Spain (Bota et al., 2016) could be exploited as a means of helping viticulture adapt in the face of future climatic challenges.

Leaf water use efficiency is also significantly affected by crop developmental stages, as was observed in the present study, with WUE being significantly higher at full bloom and berry set stages than at the 5-leaf stage and afterwards near berry ripening. A similar WUE trend has been observed in tomatoes, with significantly low WUE near maturity compared to at other stages, such as vegetative growth, flowering, fruit set, fruit growth and development stages. It has also been highlighted that from a physiological perspective fruit set is a critical growth stage (Xiukang and Yingying, 2016). Furthermore, WUE is affected by crop management practices at key developmental stages (Zhou et al., 2020); therefore, balanced crop management is needed for higher WUE efficiency.

The results of the multivariate PCA analysis revealed discrete genotypic variability among the grapevine cultivars in terms of key physiological attributes; for instance, cv. Sugraone was physiologically more responsive and efficient than the other cultivars. Sabir and Yazar (2015), Carvalho et al. (2015) and Greer (2018) have also shown that grapevine cultivars vary in terms of physiological responses under varying environmental conditions. As well as genotypic variability, environmental conditions also highly influence grapevine physiological activity; for instance, the high physiological activity observed in Islamabad in the present study can be associated with the district's colder environmental conditions compared to the warmer conditions of Chakwal, particularly in 2019. These finding are in line with those of Deus et al. (2016), Greer (2018) and Greer (2020), who also observed lower physiological activity of grapevines under elevated temperature conditions.

The varied genotypic responses of the grapevine cultivars observed in this study provide an opportunity to select cultivars with physiological adaptations under high temperature regimes. The rise in growing season temperature negatively affected all the grapevine cultivars; a site specific management strategy would thus be necessary to avoid any physiological stress occurring. The results of the present research also highlight the need to apply special management techniques during seasonal heat waves. An adaptation strategy will need to take into account the critical vine developmental stage (i.e., blooming to berry set), as indicated by the results of this research. Comprehensive short-, medium-, and long-term adaptation strategies are necessary for a sustainable viticultural industry. In this regard, short-term measures, such as delaying ripening, scheduling irrigation, applying supplemental irrigation, vine coverings and shading, and use of anti-transpirants (van Leeuwen and Destrac-Irvine, 2017; Caravia et al., 2016; Savi et al., 2018; de Palma et al., 2022) need to be further studied for their effect on vine physiology. Medium- to long-term strategies, such as considering wild rootstocks, selecting physiologically efficient cultivars, introducing characteristics from C-4 plants, and identifying new viticultural zones, could help vines adapt to future climates (Nazir et al., 2022; Rafique et al., 2023ab). Future studies that develop integrated phenology, physiology, yield and fruit quality models using dynamic approaches (Hopf et al., 2022) would be helpful to better understand the complex physiological and biochemical processes of vines.