Borage (Borago officinalis L.), from the Boraginaceae family, has industrial, pharmaceutical, and forage applications (Peiretti et al., 2004). The leaves, the flowers, and the oil extracted from the seeds are used as medicine. It contains various biologically active substances that can be used in treating diseases such as arthritis, diabetes, heart diseases, multiple sclerosis, and eczema (Asadi-Samani et al., 2014). Plant growth is a dynamic process that is continuously affected by environmental conditions (Khadem Moghadam et al., 2020; Wang et al., 2020; May et al., 2021). Based on the appearance and morphological characteristics of borage, it seems that this plant can tolerate water deficit to some extent after seedling establishment.

Plants experience several biotic and abiotic stresses during the growing season (López-Serrano et al., 2017; Jin et al., 2021; Tiwari et al., 2022). Among the different types of stresses, drought emerges as the most critical abiotic environmental stress, which causes various biochemical and physiological changes, and limits plants’ growth and production (Heshmat et al., 2021; Raza et al., 2021; Ullah & Farooq, 2021). Some adverse effects of water deficit on plants can be diminished by seed priming. Seed priming is used to speed up and synchronize the seedling emergence of many species and to produce vigorous plants with better tolerance to adverse environmental conditions (Ashraf & Foolad, 2005; Abbasi Khalaki et al., 2021). Seedling emergence and establishment may be improved by hydro- and osmo-priming treatments (Ghassemi-Golezani et al., 2008). Seed pretreatment with potassium nitrate (KNO₃) and monopotassium phosphate (KH₂PO₄) has been recommended as a low-cost and efficient way of priming seeds (Abdulrahmani et al., 2007; Ghassemi-Golezani et al., 2008).

Plants subjected to various abiotic stresses can overproduce reactive oxygen species (ROS) (Aliyari Rad et al., 2021). These species are typically the result of activating O2 to form singlet oxygen (1O2) or reducing O2 to superoxide radical (O2–•) and hydroperoxyl radical (HO₂•), and other byproducts like hydrogen peroxide (H₂O₂) or hydroxyl radical (OH•) (Tuteja et al., 2011; Halliwell & Gutteridge, 2015). A low level of ROS is essential for intracellular signaling and acclimation of plants to abiotic stresses (Suzuki & Mittler, 2006), but their production at high levels can seriously damage biological macromolecules and eventually leads to cell death (Farooq et al., 2009). Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) have a crucial role in scavenging these harmful molecules, membrane integrity preservation, and protection of DNA and proteins (Sharma et al., 2012). Changes in antioxidant activity or content reflect the environmental stress effects on plant metabolism.

Under drought stress, plants accumulate various organic and inorganic compatible solutes (osmolytes) in the cytosol to reduce the osmotic potential of the cell and maintain the cell volume and turgidity against dehydration, which is identified as osmotic adjustment (Anjum et al., 2011). Osmolytes are non-toxic compounds with low molecular weight, which do not interfere with cell metabolism and protect plants from adverse effects of stress (Farooq et al., 2009). Compounds such as proline and soluble sugars have a significant role in osmotic adjustment, and protection of membranes and macromolecules.

Light energy is absorbed by chlorophylls and carotenoids, and is transferred into the plant photosynthetic apparatus (Uvalle-Sauceda et al., 2008). Under drought stress, the level of photosynthetic pigments changes dramatically. Chlorophyll (Ch) has a key role in light trapping and converting it into chemical energy, so any decline in chlorophyll content may decrease the photosynthesis. Chlorophylls a and b are the main photoreceptors for photosynthesis (Pareek et al., 2017). Carotenoids not only contribute to light absorbance (Jaleel et al., 2009), but also protect cell membranes from ROS-induced oxidative damage (Verma & Mishra, 2005). Specifically, β-carotene can directly quench triplet chlorophyll that prevents singlet oxygen generation, inhibits lipid peroxidation, and stabilizes membranes (Farooq et al., 2009). Plant species with higher carotenoids content under oxidative stress show effective defense and better tolerance against water deficit (Farooq et al., 2009).

The capacity of plants for photochemical activity is limited. Excess solar energy absorbed (more than photochemical consumption) is released as heat in non-photochemical processes or reflected as red light known as chlorophyll a fluorescence. Measuring chlorophyll a fluorescence including initial fluorescence (F₀), maximal fluorescence (F_m), variable fluorescence (F_v), quantum yield baseline (F₀/F_m), maximum primary yield of photosystem II (F_v/F₀), and maximum quantum yield of PSII photochemistry (F_v/F_m) provide basic information about many aspects of the photosynthetic mechanism in plants (Ranjbar-Fordoei et al., 2006). The amount of F_v/F_m indicates the health of the thylakoid membrane and the relative efficiency of electron transfer from PSII to PSI under stress (Rahbarian et al., 2011). Also, F_v/F₀ is an index of the number and size of active photosynthetic reaction centers (Cen et al., 2017), and shows the photosynthetic capacity of leaves (Li et al., 2006).

Despite the fact that borage is widely cultivated in various parts of the world, little information is available about the antioxidant enzyme, compatible solutes, photosynthetic pigments and photosynthesis mechanism under limited irrigation and severe drought conditions. Therefore, the aim of this study is to examine the general response patterns of the plant to water fluctuations from a biochemical and physiological perspective, with the main target of providing reliable physiological criteria for the screening of drought-resistant genotypes in the future.

It is reported that seed priming increases borage field performance by improvement the emergence rate of seedling and leaf area index of this medicinal plant (Dastborhan & Ghassemi-Golezani, 2015). In the present study, the effect of seed priming was significant only on SOD3 and Ch a/Ch b ratio, probably due to the natural capability of this plant to reduce harmful effects of water limitation.

Based on our findings, the activity of SOD1, SOD2 and SOD3 isoforms were considerably increased in borage under moderate and severe water deficit, indicating that borage can remove the ROS under stress by SOD increment. Water deficit disturbs the equilibrium between trapping and utilization of light, which reduces photosynthetic activity in leaves. The excess energy of light in the photosynthetic system results in excessive production of ROS (Lisar et al., 2012). The capability of antioxidant enzymes to scavenge the ROS is related to plant resistance to water deficit and oxidative stress caused by it (Anjum et al., 2011). SOD isoforms are strong antioxidants that act as the first line of defense against ROS (Wang et al., 2018) and catalyze superoxide anion (O₂–•) dismutation to hydrogen peroxide (H₂O₂) in almost all plant cellular compartments. This relatively stable product can be converted to H₂O by peroxidase and catalase (Apel & Hirt, 2004). Therefore, the rising of SOD activity could enhance the plants’ ability to scavenge O₂–• radicals, thereby preventing membrane damage (Aydin et al., 2011). Excessive H₂O₂ content is toxic for cells. Therefore, rapid scavenging of H₂O₂ by the antioxidant defense system is crucial for plants (Guo et al., 2006).

Catalase activity in borage was very high under normal irrigation, while it was reduced with increasing irrigation intervals. The reduction of CAT activity under stress can be attributed to synthesis inhibition of the enzyme, its degradation by peroxisomal proteases or its photo-inactivation (Abedi & Pakniyat, 2010). The balance of CAT, ascorbate peroxidase (APX) and SOD activities is crucial for suppressing toxic ROS levels in a cell. Sometimes, an increase in activity of one antioxidant enzyme may cause a decrease in the activity of others, suggesting a delicate balance related to what these enzymes deactivate (Apel & Hirt, 2004).

Osmolytes have an essential role in plant resistance to drought by preventing water loss and maintaining cell turgidity. Changes in leaf carbohydrate status in response to stresses can act as a metabolic signal. The soluble sugars accumulation in borage plants under moderate and severe water limitation increases resistance to drought stress. Under water deficit, soluble sugars can act as osmotic agents (facilitators of osmotic adjustment) or osmo-protectors (stabilizers for the structure of proteins, enzymes, and membranes) (Marcińska et al., 2013). Some functions of plant solutes are associated with their highly hydrophilic property, and therefore may replace water molecules around membranes, proteins, and nucleic acids during drought stress. The accumulation of soluble sugars in leaves under water deficit may result from degrading the complex sugars into simple sugars and reducing their transport to the other parts such as grains (Jinyou et al., 2004).

The ability of plants to retain the biological functions of photosynthesis under water deficit conditions is an important evidence of stress tolerance. Furthermore, chlorophyll content changes during drought stress depend on the severity and duration of water limitation (Fathi & Barari, 2016). The results of the current study showed the chlorophyll content of plant leaves under drought stress has increased, which is consistent with the findings of some researchers (Teixeira & Pereira, 2007; Azhar et al., 2011). The increment in chlorophyll content under stress conditions could help the plants to cope with environmental stress and compensate for any damage that could affect the integrity and function of the photosynthetic system. The increase in chlorophyll content due to decreasing water supply may be associated with a reduction in leaf area and relative water content (Dastborhan & Ghassemi-Golezani, 2015), and an increase in leaf thickness and chloroplast density in stressed borage plants. This could be a defensive response to decrease the adverse effects of drought stress (Farooq et al., 2009). On the other hand, chlorophyll and proline are both synthesized from the common constituent of glutamate. In most plants that are under water deficit, the synthesis of chlorophyll is limited due to increasing conversion of glutamate to proline by the enzyme of gamma-glutamyl kinase (Handa et al., 1986). Since proline accumulation in borage plants under the studied irrigation treatments was not significant compared to normal irrigation, glutamate may be used in the synthesis of chlorophyll. The decrement in chlorophyll a/b ratio under water limitation (Table 7) is due to the larger increase of chlorophyll b compared to chlorophyll a in stressed plants. Sharifi & Mohammadkhani (2016) also found a reduction in the Chl a/Chl b ratio in wheat genotypes under water deficit conditions.

The results of our work revealed that irrigation up to 120 mm evaporation did not cause any change in leaf carotenoids content, but severe water stress acted as a stimulus to produce more carotenoids in borage leaves. Plants can protect cell structures against active radicals produced under stress through the production of antioxidant compounds such as carotenoids (Bettaieb-Rebey et al., 2011). Carotenoids are isoprenoid compounds that contribute not only to light absorbance and passing of solar energy to chlorophyll but also to helping plants to withstand water limitation (Jaleel et al., 2009) and to protect chlorophyll from photodamage. Carotenoids are among the most potent phytochemicals because of their properties as antioxidants and their role in the protection of reaction centers and wasting extra light energy (Shah et al., 2017). Plant species with a higher content of carotenoids under oxidative stress show effective defense and better tolerance against drought stress (Farooq et al., 2009).

In the current study, the enhancement of F₀ and reduction of F_m under drought stress led to an increment in F₀/F_m and a decrement in F_v/F₀ and F_v/F_m concurrently. Chlorophyll a fluorescence is a simple, rapid, cheap, and non-destructive tool for evaluating light-dependent photosynthetic reactions and screening genotypes for water deficit tolerance (Kalaji et al., 2016). Water deficit influences F_v/F_m and reduces the rate and capacity of electron transport. Therefore, the system reaches the F_m state faster and leads to a reduction in the F_v state. In the F₀ state, plastoquinone electron acceptor pool (QA) is at its complete oxidation level (Li et al., 2006). The increment in F₀ can damage the photosynthetic system (Yamane et al., 1997). The reduction of F_m due to drought stress could be related to the increment of non-photochemical dissipation (as heat) or decrement in the activity of enzyme complex that catalyzes the water-splitting (Lucena et al., 2012).

In the present study, F₀/F_m significantly increased as a result of water shortage. The higher F₀/F_m under drought stress shows that the initial reduction rate of the plastoquinone A (PQA) was more than its re-oxidation rate by the plastoquinone B (PQB) and PSI activity (Lucena et al., 2012) when the plants were irrigated with longer irrigation interval.

The F_v/F₀ decrease in borage under water deficit (Table 6) could be due to a disorder in the photosynthetic apparatus and a reduction in the number of reaction centers. F_v/F₀ ratio was negatively correlated with the chlorophyll content index under water deficit (Akhkha, 2009).

The F_v/F_m ratio can be used to detect damages to PSII and possible photo-inhibition. Decreasing F_v/F_m under drought stress (Table 4) revealed that the PSII might be damaged in different levels due to the water deficit effect (Li et al., 2006). Drought stress through its adverse impacts on the CO₂ entry reduces the capacity of reception and electron transport. As a result, fluorescence is rapidly maximized, leading to a decrement in F_v. Indeed, limitation of CO₂ absorption due to stomatal closure under water deficit disturbs the equilibrium between the photochemical activity of PSII and the photosynthesis electron need and damages the centers of PSII (Zlatev & Lidon, 2012).

In conclusion, moderate and severe water stresses led to an increment in SOD1, SOD2 and SOD3 activities, soluble sugars, and initial fluorescence. The mean contents of Ch a, Ch b, and Ch a + Ch b under mild, moderate and severe water deficit were significantly higher than that of normal irrigation. The highest carotenoids content and quantum yield baseline parameter (F₀/F_m) of borage leaves were obtained under severe drought stress. However, water limitation decreased the Chl a/Chl b ratio, F_v/F₀ and F_v/F_m in borage leaves. It seems that the improvement in SOD activity and increasing soluble sugars and carotenoids accumulation have a decisive role in enhancing drought tolerance in borage plants. Seed pretreatment had no meaningful effect on the activity of antioxidant enzymes, accumulation of compatible solutes, photosynthetic pigments content and most of the fluorescence parameters in borage leaves.