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Environmental sustainability of viticulture is negatively affected by prolonged droughts. In limestone dominated regions, there is limited knowledge on grapevine water status and on methods for accurate evaluation of actual water demand, necessary to appropriately manage irrigation. During a dry vintage, we monitored plant and soil water relations in old and young vines of Istrian Malvasia on Karst red soil. The vineyard with young vines was additionally subdivided into two areas, based on their soil type, 1) karst silty-clay loam, and 2) mixture of crushed rocks and karst silty-clay loam (stony soil). Seasonal changes in exploited water resources were estimated via analysis of oxygen isotope composition (δ18O) of rainfall, deep soil water, and xylem sap. We hypothesized that plants are able to thrive during drought thanks to the water stored in deep soil layers, while they rely less on superficial soil horizons. Our results show that vines growing on karstic substrates have deep roots securing the use of stable water sources during summer, with consequent favourable plant water status. In fact, both young and mature vines approached the threshold of severe water stress, but never surpassed it, as midday leaf water potentials were >−1.3 MPa in all study sites. Vines roots showed flexible water uptake, i.e. the ability to absorb water from deep or shallow soil horizons during drought and after late-summer thunderstorms, which was particularly evident in vines growing on the stony soil. In fact, precipitations of 20 mm were enough for plant water status recovery, due to fast infiltration. On the other hand, at least 50 mm of rainfall were necessary to induce water status recovery in more compact soil (karst silty-clay loam). Our findings provide new knowledge on the rooting depth and water needs of vines growing on shallow soils overlying fractured limestone bedrock. Unlabelled Image • Vines' water requirements and sourcing on limestone soils are not well understood. • Deep roots ensured the uptake of stable water resources during summer drought. • Stony soil favoured fast water infiltration and its absorption by vines roots. • Roots of young vines were sufficiently developed four years after planting. • Pre-dawn water potential estimates accurately plants water needs in karstic areas. [ABSTRACT FROM AUTHOR]