Physiological traits determining high adaptation potential of sweet briar (Rosa rubiginosa L.) at early stage of growth to dry lands

Little is known about mechanisms of sweet briar adaptation to dry habitats. The species is highly invasive and displaces native plants from dry lands of the southern hemisphere. This study evaluates physiological basis of Rosa rubiginosa L. adaptation to soil drought. We performed a pot soil drought experiment and assessed water relations, water use efficiency, gas exchange and photosynthetic apparatus activity. The study also measured the content of chlorophyll, soluble carbohydrates and proline and analyzed plant biomass growth. We hypothesized that the drought stress induced an effective mechanism enabling adaptation of young sweet briar roses to soil water deficit. The study identified several adaptation mechanisms of R. rubiginosa allowing the plant to survive soil drought. These included limiting transpiration and stomatal conductance, increasing the level of soluble sugars, reducing chlorophyll content, accumulating CO2 in intercellular spaces, and increasing the quantum yield of electron transport from QA− to the PSI end electron acceptors. As a result, young sweet briar roses limited water loss and photoinhibition damage to the photosynthetic apparatus, which translated into consumption of soluble sugars for growth purposes. This study showed that photosynthesis optimization and increased activity of the photosynthetic apparatus made it possible to avoid photoinhibition and to effectively use water and sugars to maintain growth during water stress. This mechanism is probably responsible for the invasive nature of R. rubiginosa and its huge potential to displace native plant species from dry habitats of the southern hemisphere.

Rosa rubiginosa L., known also as sweet briar, belongs to the group of wild growing roses and is a species native to the northern hemisphere. It was spread, naturally or through anthropogenic and animal participation into the southern hemisphere [1][2][3] . Sweet briar rose was introduced to South America, Australia and New Zealand, where it adapted to the new environment and occupied such areas as forests, and dry lands such as steppes. In South America, its wild growing shrubs can be found predominantly in Chile and Argentina 2,4 . It is a fast-growing shrub with a bushy and effusing habit that prefers habitats exposed to sunlight but may also grow in partial shade 5 .
In many regions of the southern hemisphere sweet briar rose is treated as a highly expansive weed. Abundance of herbivorous fauna in the southern hemisphere, for which the fruits of sweet briar are valuable forage, is an additional factor of this species propensity to expansion 6 . Plant achenes undergo scarification in the animal digestive tract, which after their defecation accelerates the seed germination. From the ecological perspective, invasive varieties of sweet briar rose may pose a threat to the ecosystem they grow in since they contribute to the displacement and extinction of valuable species of indigenous plants.
R. rubiginosa was introduced to New Zealand in 1800 and has since been treated as a truly parasitic species 7 . In New South Wales (Australia), since 1919, it has been treated as one of 20 most persistent weeds 8 . In 1960, New Zealand tried to apply biological methods to control invasive varieties of sweet briar rose. To reduce the population of this shrub, an attempt was made to use rose-seed megastigmus, Megastigmus aculeatus (of hymenoptera

Results
On the thirtieth day of drought (D), soil water potential (Ψ W-S ) reached ca. −0.75 MPa and was significantly lower than in the optimally watered variant (−0.006 MPa) (C) (Fig. 1A). Soil water deficit induced water stress in the leaves of sweet briar manifested in reduced water potential (for C: Ψ W = −0. 16  Leaf dehydration caused significant reduction of photosynthesis (A N ) ( Fig. 2A) and transpiration rate (E) (Fig. 2B), as well as stomatal conductance (g S ) (Fig. 2C). On the thirtieth day of water deficit, a clear increase in intercellular concentration of CO 2 (C i ) was noted (Fig. 2D).
At the leaf level, we saw a significant reduction in water use efficiency (WUE), calculated as a ratio of photosynthesis rate and transpiration rate, i.e. so called instantaneous WUE (Fig. 3A). A similar drop was confirmed for so called intrinsic WUE, i.e. a ratio of A N to stomatal conductance (Fig. 3B). However, at the whole plant level WUE values increased during soil drought, as showed in the ratio of plant biomass growth and the amount of water provided (Fig. 3C).
Drought enhanced the levels of soluble carbohydrates (SC) both in the roots and leaves of sweet briar (Fig. 4A). Their levels in the leaves were higher than in the roots for both control and drought conditions. After 30 days of drought, only roots exhibited a significant increase in proline content (Fig. 4B). In dehydrated leaves, its level was similar as in those optimally hydrated.
Limited water content determined for the thirtieth day of the study considerably reduced fresh (FW) and dry weight (DW) of above ground parts and roots (Fig. 5A). Soil drought limited also elongation growth of sweet briar, clearly manifested in lower plant height (Fig. 5B). Also, the root system of drought exposed plants was much less developed than in controls (Fig. 5B).
On the thirtieth day of drought the photosynthetic apparatus of sweet briar showed higher activity than in optimally hydrated plants (Table 1). Significant changes occurred for a majority of the analyzed parameters of chlorophyll fluorescence except for ABS/CS m that involves the amount of energy absorbed by antennas (1922 for control, 1977 for drought, 103.9% of control).
Chlorophyll fluorescence parameters recorded during water stress indicated enhanced efficiency of excitation energy use (reduction in DI o /CS m -87% of control, increase in ET o /CS m -133% of control, increase in TR o /CS m -110% of control). Chlorophyll fluorescence measurements revealed also a rise in quantum yield of electron transport (ϕR o , 148% of control) from Q A − to the end acceptors in PSI. The changes in the photosynthetic activity recorded on the thirtieth day of drought were accompanied by significant drop in chlorophyll content (Table 1).

Discussion
During their evolution, plants have developed adaptation mechanisms to habitats of low soil water content 19,20 . Those plants include species with high potential for colonization of dry sites. One of these is R. rubiginosa, the invasive populations of which conquered dry areas of the southern hemisphere 3 . Its high ability to thrive at unfavorable soil moisture level may be due to developing specific and effective adaptation mechanisms. This is particularly important during interaction of young plants of low biomass (one year old plants in our experiment) with intense stress represented here by soil drought (in our experiment involving 30 days of limited irrigation).
Common responses to leaf dehydration include reduction of photosynthesis, transpiration and partial closure of stomata 21,22 . These are adaptive responses aimed at limiting water loss but they also inhibit plant growth 23,24 .
Sweet briar is a C 3 plant 25 . In our pot experiment, limited watering induced water stress (Fig. 1B), manifested in reduced photosynthetic activity ( Fig. 2A), limited transpiration ( Fig. 2B) and partial closing of the stomata (Fig. 2C). Despite limited gas exchange R. rubiginosa still accumulated CO 2 (C i ) in its intercellular spaces (Fig. 2D). Enhanced C i occurs mainly in plants with CAM photosynthesis type 26 . At limited stomatal conductance an increase in CO 2 concentration may be an alternative for photosynthesis optimization in the investigated species during leaf dehydration. Studies on water stress in C 3 plants usually indicate lowering of intercellular concentration of CO 2 27-29 . The accumulation of CO 2 by R. rubiginosa makes the plant somewhat independent of atmospheric CO 2 , the availability of which is limited by partial close of the stomata. Another reason may involve (2019) 9:19390 | https://doi.org/10.1038/s41598-019-56060-3 www.nature.com/scientificreports www.nature.com/scientificreports/ changes in the internal leaf structure caused by cell dehydration and shrinking. Hura et al. 30 showed that leaf dehydration in sweet briar exposed to salt stress causes cell shrinking and widens intercellular space available for CO 2 accumulation.
High C i values were accompanied by intensified activity of the photosynthetic apparatus and reduced chlorophyll content (Table 1). Lowering chlorophyll content under water stress is one of the ways to avoid photoinhibition damage to the photosynthetic apparatus 31-34 . Kyparissis et al. 35 found that summer survival of Phlomis www.nature.com/scientificreports www.nature.com/scientificreports/ fruticosa shrub under Mediterranean climate depended on avoidance of photoinhibition damage through decreasing chlorophyll content. Water stress makes plants more susceptible to damage inflicted by excessive UV-Vis radiation 36 .
In our opinion, the increase of intercellular CO 2 in dehydrated plants is a means for photoinhibition prevention by providing a sink for electron transport. This statement seems supported by high values of ϕR o ( Table 1) indicating increase of the quantum yield of electron transport from Q A − to the PSI end electron acceptors 37  www.nature.com/scientificreports www.nature.com/scientificreports/ NADPH 2 generation that together with ATP is used in the dark reaction to reduce CO 2 to carbohydrates 38 . The relationship between high values of C i and photoprotection in R. rubiginosa may be concluded from a significant increase in F v /F m ratio (Table 1)  www.nature.com/scientificreports www.nature.com/scientificreports/ suggests that higher values of C i in drought treated plants guarantee a sufficient sink for electrons, thereby decreasing the need for energy dissipation (low values of DI o /CS m ) ( Table 1).
Increased activity of the photosynthetic apparatus and photosynthesis optimization under leaf dehydration enhanced the content of soluble carbohydrates in both roots and leaves (Fig. 4A). Soluble carbohydrates may be utilized during plant growth 39 , but as osmotically active substances they also retain water in the cell and limit its dehydration 40 . Proline is another substance with osmoregulatory function but in our study its increased accumulation in the roots (Fig. 4B) is probably associated with its signaling role in the expression of drought resistance genes 41 . conclusions Sweet briar drought adaptation mechanisms outlined above include efficient water managements at the entire plant level (WUE WP ) (Fig. 3C). Considering the study outcomes, we suggest that R. rubiginosa response to soil drought involves the adaptation mechanisms resulting not only in water deficit tolerance but also ensuring effective water management (Fig. 6). These mechanisms include limiting transpiration and stomatal conductance, increasing the level of soluble sugars, reducing chlorophyll content, accumulating CO 2 in intercellular spaces, and increasing the quantum yield of electron transport from Q A − to the PSI end electron acceptors. As a result, sweet briar exposed to drought stress is capable of limiting both water loss and photoinhibition damage to the photosynthetic apparatus. www.nature.com/scientificreports www.nature.com/scientificreports/ Moreover, photosynthesis optimization during water stress triggered consumption of soluble carbohydrates for growth processes. This should be attributed to huge invasive potential of R. rubiginosa, whose growth dynamics in dry environment needs to be competitive to native species of the southern hemisphere.

Material and Methods
plants material and growth conditions. The study involved 13 month old sweet briar roses after their first overwintering, and with fully lignified stems. After stratification, the germinating seeds were sown into pots Drought conditions. Soil drought was applied by limiting watering for 30 days -from 8 May to 6 June 2018.
Optimally watered plants received in total 9 L of water per pot, while those exposed to drought only 2.7 L, i.e. 70% less. Irrigation frequency and the amount of water depended on the temperature inside the garden tunnel. For the last three days (28-30) of soil drought the plants did not receive any water.   Table 1. Changes in 10 chlorophyll fluorescence parameters and chlorophyll (Chl) content on the thirtieth day of soil drought. C -control, D -drought, % of C -D values exhibited as percentage of control. Mean values ± SE (n = 10). Asterisks at individual parameters mark differences significant at p < 0.05 vs. control, Student's t-test.

R -F W R -D W A G P -F W
Gas exchange. Photosynthesis rate (A N ), transpiration rate (E), stomatal conductance (g S ), and intercellular concentration of CO 2 (C i ) were measured using an infrared gas analyzer LCpro-SD (ADC BioScientific Ltd., UK). The conditions in the measurement chamber were as follows: carbon dioxide concentration 360 µmol CO 2 mol −1 air, air humidity equal to ambient humidity, temperature 28 °C, PAR intensity 600 µmol photons m −2 s −1 .
The measurements were carried out between 10:00 a.m. and 12:00 p.m. and involved the first three leaflets of the largest assimilation area.
Water use efficiency. Additionally, we determined an instantaneous water use efficiency index (WUE) representing A N /E ratio, and intrinsic WUE representing A N /g S ratio. WUE of the whole plant (WP) was estimated as follows:  Statistical analysis. Statistical analysis was carried out using Statistica v. 12 (Statsoft Inc., Tulsa, OK, USA).
Analysis of variance was used to determine the main effects of drought stress on physiological and biochemical parameters. Before ANOVA, data were checked for normality and homogeneity of variance. The Duncan's multiple range test at the probability level of 0.05 was performed to estimate the significance of differences between treatment means. Differences between two means were compared by the Student's t-test.