1Water stress and increasing temperatures are two main constraints faced by plants in the context of climate change. These constraints affect plant physiology and morphology, including phenology, floral traits, and nectar rewards, thus altering plant–pollinator interactions.

2. We compared the abiotic stress responses of two bee-pollinated Boraginaceae species, Echium plantagineum, an annual, and E. vulgare, a biennial. Plants were grown for 5 weeks during their flowering period under two watering regimes (well-watered and water-stressed) and three temperature regimes (21, 24, 27°C).

3. We measured physiological traits linked to photosynthesis (chlorophyll content, stomatal conductance, and water use efficiency), and vegetative (leaf number and growth rate) and floral (e.g., flower number, phenology, floral morphology, and nectar production) traits.

4. The physiological and morphological traits of both species were affected by the water and temperature stresses, although the effects were greater for the annual species. Both stresses negatively affected floral traits, accelerating flower phenology, decreasing flower size, and, for the annual species, decreasing nectar rewards. In both species, the number of flowers was reduced by 22–45% under water stress, limiting the total amount of floral rewards.

5. Under water stress and increasing temperatures, which mimic the effects of climate change, floral traits and resources of bee-pollinated species are affected and can lead to disruptions of pollination and reproductive success.

Growth conditions

Seeds were provided by Semailles nursery (Faulx-les-Tombes, Belgium). Seeds were placed in a germination chamber (Economic Delux model ECD01E; Snijders Scientific, Tilburg, The Netherlands) under 20°C/18°C day/night temperature and a 16-h light (L):8-h dark (D) photoperiod, for 2 weeks. Seedlings were transplanted into pots filled with a 1:1 (v/v) mix of sand (0/5, M Pro, The Netherlands) and universal peat compost (DCM, Amsterdam, The Netherlands). Plants were grown in the greenhouse at the university campus (Louvain-la-Neuve 50°39′58′′N; 4°37′9′′E, Belgium) and were watered every 2–3 days with rainwater. Treatments were applied after floral transition under controlled conditions in growth chambers (SEFY platform, Louvain-la-Neuve) at different temperature and watering regimes.

To observe the effects of temperature and water stress (and their interaction) on vegetative and reproductive development and photosynthesis-related parameters, fifteen plants per treatment and species were placed under three temperature regimes (21/19°C, 24/22°C, and 27/25°C day/night) and two watering regimes (well-watered compared to water-stressed). The well-watered plants received daily watering (soil humidity about 25%, as determined using a Procheck Hand-held Sensor 10 HS moisture sensor, Decagon Devises, Inc, Pullman WA, USA), whereas the water-stressed plants were watered twice a week (soil humidity of 8–15%). The combination of temperature and watering regimes resulted in six treatments: 21°C well-watered (21WW), 21°C water-stressed (21WS), 24°C well-watered (24WW), 24°C water-stressed (24WS), 27°C well-watered (27WW), and 27°C water-stressed (27WS). In total, 90 plants per species were monitored in three growth chambers. The photoperiod was set to 16L:8D, and relative humidity was maintained at 80 ± 10%. Growth chamber experiments lasted for 6 weeks. Water stress was applied after 1 week of acclimation to the growth chambers; this initial week was considered week 0.

Morphological traits

At week 0, flowering stem height was measured. Every week for 6 weeks, the number of axillary stems (for E. plantagineum), new leaves (>2 cm), inflorescences, and flowers at anthesis were counted per plant. At the end of the experiment (week 5), the height of the main flowering stem was measured to calculate the growth rate.

Physiological traits

The 5th-node leaves of 10 plants per treatment were measured at the beginning of the experiment and 2 weeks after inducing stress. The chlorophyll content index (CCI) was measured using a chlorophyllometer (Opti-Sciences, CCM-200), and three measurements were taken per leaf. An automatic porometer (AP4 System, Delta-T Devices) was used to measure the stomatal conductance. Gas exchange was measured using an infrared gas analyzer (IRGA ADC BioScientific LCI-SD system, serial No.33413, Hoddesdon, UK). The instantaneous water use efficiency (WUE_i) was calculated as WUE_i = A_i/E_i.

Floral and nectar traits

The corolla depth and diameter were measured three times, at weeks 1, 3, and 5, on 10 random flowers in each treatment. In week 3, flowers were dissected, and floral organs were scanned (Ricoh MP C3004 ex PS). The corolla surface area and the length of all stamens per flower were calculated using ImageJ software.

Nectar was extracted with glass capillary tubes (1, 5, or 10 μl, depending on the nectar volume; Hirschmann Laborgeräte, Eberstadt, Germany) from five flowers per treatment (from five different plants). Total sugar concentration (°Brix) was measured with a low-volume hand refractometer (Eclipse hand-held refractometer; Bellingham and Stanley, Tunbridge Wells, UK). Nectar sugar content per flower (mg) was calculated following Prys-Jones and Corbet method (1991).