Among all the physiological processes that are essential to plant growth and production, photosynthesis is one of the most affected traits under drought (Cameron et al., 1989; Farquhar et al., 1989; Condon et al., 2004; Rho et al., 2012) ( Figure 2 ). Photosynthesis is composed of several interconnected physical and biochemical processes that result in CO₂ fixation in mesophyll cells followed by the accumulation of sugars, cellular components, and biomass. The light and CO₂ assimilation reactions are impacted by drought via different mechanisms including 1) biophysical limitations caused by stomatal closure (Cornic, 2000) and limited CO₂ diffusion through the mesophyll (Sharkey et al., 2007), and 2) biochemical limitations such as reductions in the electron transport chain in the thylakoid (Estrada et al., 2015; Pilon et al., 2018) or decreased activity of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Flexas and Medrano, 2002; Lawlor and Cornic, 2002; Flexas et al., 2006; Morales et al., 2020).

Blueberries can show physiological changes as soon as three to four days after drought stress occurs (Améglio et al., 2000). First, leaf and stem water potential will quickly drop in the presence of water deficiency (Davies and Johnson, 1982; Améglio et al., 2000). Decrease in water potential will then trigger rapid closure of stomata to reduce transpiration and therefore limit water loss (Améglio et al., 2000; Perrier et al., 2000; Mingeau et al., 2001). Reduced stomatal conductance can help plants to maintain the minimum water potential at a stable level and therefore avoid severe damage such as xylem embolism (Améglio et al., 2000). As a result of stomatal closure, the photosynthetic rate tends to decline due to decreased intercellular CO₂ concentration and other changes (e.g., reduced chlorophyll content) caused by drought (Sade et al., 2012). In blueberry, this quick stomatal closure is mediated by increases in the abscisic acid sent from the roots to the shoots (Farooq et al., 2012; Chen et al., 2017). In an experiment performed in SHB blueberries, Chen et al. (2017) observed that as drought progressed during a 16-days period, photosynthesis decreased due to stomatal closure. Decrease in photosynthesis and stomatal conductance in blueberries under drought stress has also been reported in multiple studies based on measuring leaf photosynthesis using a leaf infrared gas analyzer (LI-6400/XT Portable Photosynthesis System, Lincoln, NE, United States: LI-COR Biosciences; Cameron et al., 1989; Améglio et al., 2000; Chen et al., 2017).

Carbon isotope discrimination (Δ¹³C) is another effective way to reflect how drought reduces stomatal conductance and photosynthesis, in addition to directly measuring stomatal conductance and photosynthetic capacity. Three forms of carbon isotopes are present in the atmosphere with ¹²C and ¹³C being the stable isotopes and ¹⁴C a radioactive isotope (Graven et al., 2020). For the stable isotopes, the light isotope ¹²C is more abundant than the heavy stable isotope, ¹³C. In plants, ¹²C is preferably fixed by Rubisco during photosynthesis because of its abundance and smaller size in comparison with ¹³C (Farquhar et al., 1989). Under drought-triggered stomatal closure, there is less opportunity for ¹³C to diffuse out of the stomatal cavity leading to greater assimilation of this isotope, and higher concentrations of ¹³C in plant dry matter (Farquhar et al., 1982). Δ¹³C is defined as the deviation of isotope effects (α) from the unity, which equals α −1 = R_r /R_p −1, where R_r is the ¹³C/¹²C molar ratio of reactant and Rp is the molar ratio of products (Farquhar et al., 1989). In general, when plants fix more ¹³C, there is less C¹³ discrimination and therefore a lower Δ¹³C. Δ¹³C has been used widely to study the effects of drought on stomatal closure for its effectiveness in reflecting stomatal behavior during the whole growing season, which is powerful especially when gas exchange measurements are not feasible (Farquhar et al., 1989; Condon et al., 2004). In blueberry, Balboa et al. (2020) showed that drought decreased the Δ¹³C values in all studied cultivars. These authors observed that some cultivars such as Brigitta and Biloxi had lower Δ¹³C than Sharpblue. As low Δ¹³C has been related with high water use efficiency and drought tolerance, the Δ¹³C discrimination technique could be used to screen for these kinds of traits in the future (Condon et al., 2004; Zhang et al., 2022; see drought tolerance section).

Following drought-induced stomatal closure, photorespiration can be triggered due to the combination of low intercellular CO₂, low transpiration rate, and high leaf temperature ( Figure 2 ). During photorespiration, the enzyme Rubisco will oxygenate ribulose-1,5-bisphosphate (RuBP) to produce glycerate-3-phosphate, and 2-phosphoglycolate. As a result, photorespiration decreases the efficiency of photosynthesis and produces reactive oxygen species (ROS) such as such as O₂ ^-, O₂ ¹, H₂O₂, RO and OH^- that can damage membranes and enzymes (Chastain et al., 2014; Gahir et al., 2021). In addition to photorespiration, reduced CO₂ fixation under drought also leads to an accumulation of photons as the plant is still harvesting light regardless of lower photosynthetic rate to utilize the light energy. Excessive photons accumulated in drought-stressed plants will give rise to photoinhibition ( Figure 2 ), which, according to Long et al. (1994), is “the light independent and slowly reversible retardation of photosynthesis, independent of any developmental change”. Photoinhibition is a mechanism to protect plants from light damage to photosystem II (PSII) (Long et al., 1994). In addition, photoinhibition can also decrease photosynthesis efficiency and produces ROS, similar to photorespiration (Krause, 1988). ROS can damage membrane structures and proteins in the thylakoids and therefore reduce the light reactions of photosynthesis (Maxwell and Johnson, 2000; Chastain et al., 2014; Pilon et al., 2018; Liang et al., 2019).

The efficiency of light reactions can be measured with chlorophyll fluorescence analysis. Light energy absorbed by chlorophyll can be used to drive photosynthesis reactions, lost as heat, or re-emitted as light (Maxwell and Johnson, 2000). Chlorophyll fluorescence measures how much light energy is re-emitted as light by chlorophyll, which can provide insight into the efficiency of photochemistry (Maxwell and Johnson, 2000). In blueberries, as water stress advances and the leaf water potential decreases, the light reactions also get damaged and fluoresce parameters such as quantum yield of PS II (ϕPSII) and photosynthetic electron transport rate (ETR) are significantly reduced (Estrada et al., 2015; Balboa et al., 2020). Estrada et al. (2015) and Balboa et al. (2020) observed differences in fluorescence parameters among various cultivars under drought, which suggests the potential to select cultivars based on drought-resistant photochemistry (see drought tolerance section). In addition to blueberries, drought impacts on fluorescence parameters were also observed in crops such as cotton (Chastain et al., 2014), peanut (Pilon et al., 2018), lettuce (Shin et al., 2021), and tomato (Liang et al., 2020). In lettuce seedlings, drought stress impacted chlorophyll fluorescence parameters especially under severe drought (Shin et al., 2021). In tomatoes, chlorophyll fluorescence parameters like the maximum quantum yield (F_V/F_M) and ϕPSII declined as drought stress increased, indicating closure of the PSII reaction center and reduced electron transfer (Liang et al., 2020). Although chlorophyll fluorescence provides an estimate of photosynthetic conditions, it is best combined with other techniques like gas exchange measurements because chlorophyll fluorescence does not directly measure carbon fixation (Maxwell and Johnson, 2000).

In many cases the decrease in fluoresce parameters (Estrada et al., 2015; Balboa et al., 2020) is also related to the decrease in chlorophyll and carotenoid content during drought (Chen et al., 2017; Mazurek et al., 2021; Molnar et al., 2022). Additionally, membrane damage as result of ROS accumulation can also be monitored through malondialdehyde (MDA) content, which is a product of membrane lipid peroxidation because of membrane damage (Ma et al., 2015). In a study of highbush blueberry seedlings, Chen et al. (2017) observed that drought stress increased the levels of MDA, which is likely due to membrane damage and lipid peroxidation. Membrane damage under drought can also be studied by measuring the relative electrolyte conductivity (REC) which signals electrolyte leakage due to pores in the membranes (Demidchik et al., 2014). As drought advances in blueberries, an increase in relative electrolyte conductivity can be observed, together with increased MDA and decreased ϕPSII and chlorophyll content (Chen et al., 2017).

Plants have evolved a complex antioxidant system to protect cells from ROS damages by producing enzymatic and non-enzymatic antioxidants to act as ROS scavengers (Farooq et al, 2012). Examples of enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and non-enzymatic antioxidants include polyamine (PA), salicylates, and compatible solutes including proline, and glycinebetaine (GB) (Farooq et al, 2012; Chen et al., 2017). An increase in the activities of SOD and POD was reported in blueberries under drought stress (Panta et al., 2001; Wu et al., 2016; Chen et al., 2017). In the case of SOD there was significant cultivar response in this parameter to drought, which could therefore indicate that cultivars with higher SOD could provide drought tolerance as they have more ROS scavenger capacity than other (Wu et al., 2016; see drought tolerance section).

When drought continues for a long period of time, the leaf water potential needs to remain low so that water can be drawn into the leaf from the root and maintain turgor. To do this, plants concentrate osmolytes that can be organic (soluble sugars, organic acids, and amino acids) and inorganic (ions such as Na⁺, K⁺, Ca2⁺, and Cl^–) (Farooq et al., 2012). It has been demonstrated in blueberries that as drought progresses, leaves accumulate active osmolytes such as proline (Balboa et al., 2020) and soluble sugars (Chen et al., 2017) to decrease the osmotic potential and thus the leaf water potential.

Having a prolific root system is considered a major mechanism for avoiding drought by the uptake of more water from the soil (Erb, 1993; Farooq et al., 2012). Yet, the role of root architecture and physiology in drought tolerance is not well understood in blueberry as most studies focused on the shoot instead of root response to drought. Most, if not all, of the previous studies used container-grown plants in greenhouses or rain-out shelters for the ease of managing drought stress levels. However, a plant’s ability to absorb water in deeper layers of the soil through a large or deep root system cannot be well evaluated when roots are confined in a limited space. For example, rabbiteye blueberries are known for their deeper and tougher root systems and better drought tolerance in field conditions compared to other cultivated ecotypes (Davies and Johnson, 1982; Erb, 1993). However, previous studies focusing mainly on shoot physiological responses were not able to reveal true differences in drought tolerance between RE and SHB blueberries (Estrada et al., 2015; Zhang et al., 2022). Compared to RE cultivars, SHB stems showed stronger mechanical support and safer water transport structures, despite a lower hydraulic conductivity (Zhang et al., 2022). On the other hand, a plant’s ability to uptake water from the soil to alleviate drought stress was omitted in previous studies and therefore the full picture of blueberry drought tolerance is yet to be understood.

Among the few studies investigating the response of blueberry root systems to drought, only root to shoot ratio was studied and contrasting observations were reported. Davies and Johnson (1982) studied drought response in the RE cultivar Bluegem and did not find significant changes in root to shoot ratio under water stress (Davies and Johnson, 1982). Alternatively, Cameron et al. (1989) found that two NHB cultivars Jersey and Bluecrop had a higher root to shoot ratio under severe water stress than well-watered conditions and that Bluecrop partitioned a higher percentage of dry weight to shoots compared to Jersey. The lack of studies evaluating root mechanistic responses to drought is likely due to difficulties in phenotyping roots and the challenges associated with evaluating drought tolerance in field-grown plants as compared to container-grown plants.

In the future, it would be beneficial to investigate the physiological responses of roots to drought and the distribution of roots related to drought tolerance. This could be done, for example, by screening container- or field-grown plants treated with water stress and by studying root distribution and other traits using nondestructive (e.g., X-Ray tomography, mini-rhizotron systems) or destructive approaches (digging roots out using soil core or standard excavation method) (Kuijken et al., 2015; Wasaya et al., 2018). High-throughput phenotyping tools for root image analysis will be needed to improve the efficiency of manual root phenotyping, which can be very labor intensive especially for large datasets (Kuijken et al., 2015; Wasaya et al., 2018; Atkinson et al., 2019). Additionally, integrating soil moisture sensing technologies, such as time domain reflectometry or neutron moisture meters, would facilitate measurements of volumetric water content at various depths over time, providing insight into root physiological responses to dynamic soil moisture conditions (Zhu et al., 2019). High spatial-temporal soil moisture data will help to estimate the water content available to blueberry roots under drought and water uptake by plants during a growing season (Evett et al., 2003; Sprenger et al., 2017).

Most studies on blueberry drought tolerance focused on shoot physiology without discussing fruit-related preharvest or postharvest traits. For a fruit crop like blueberry, quality and quantity of the final product should be important factors when determining cultivar performance. Evaluation of fruit-related traits for drought tolerance will require more careful experimental planning and larger space compared to studies only focusing on vegetative traits. Only mature plants during the reproductive stage can be used for fruit trait screening which will likely take more space than smaller plants used in some of the vegetative studies. It is also important to consider flowering time variation among cultivars to better plan drought treatments. Artificial chilling may be needed to supplement chilling requirement of some varieties and synchronize flowering time across cultivars to allow the application of drought treatment during the same development stage. Additionally, employment of frost protection using high-tunnels or overhead irrigation is important to protect flower or fruit damage from spring frost events in the field.

As discussed under section four–variation in drought tolerance among cultivated blueberries, only a few papers reported cultivar evaluation for drought tolerance. Among the reported studies, most focused on evaluating physiological, biochemical, or molecular responses to drought with limited discussion on the identification of drought-tolerant cultivars. For example, Cameron et al., 1989 compared physiological responses of NHB cultivars Jersey and Bluecrop under drought, which showed that Jersey was more sensitive than Bluecrop for photosynthesis but did not differ from Bluecrop for stomatal conductance or water use efficiency (Cameron et al., 1989). A comparison between seedlings of 3 NHB (Bluegold, Brigitta, Elliott) and 3 SHB cultivars (Biloxi, O’Neal, Sharpblue) suggested that Bioloxi was the most drought tolerance based on an estimated water deficit resistant index (WDRI) (Balboa et al, 2020). However, the reliance on immature plants and therefore insufficient assessment of fruit-related traits in Balboa et al. (2020) did not provide adequate evidence to connect physiological parameters with drought tolerance considering fruit quality and yield. In general, there is a lack of large-scale and comprehensive cultivar evaluation for drought tolerance, which hinders the development of drought tolerant blueberry cultivars.

Large-scale drought tolerance evaluation faces many challenges for setting up the controlled drought treatments. First, sufficient greenhouse or rain-out shelter space is needed to allow controlled irrigation, which might not be available for many programs. When rain-out shelters are not available for field trials, no irrigation field trials might be used as an alternative for regions without frequent precipitation during the growing period. However, drought stress levels will be subject to interference of rain, storms, and other weather events. Second, if cultivars with various chilling requirements are screened in the same experiment (e.g., NHB and SHB), cooler space might be necessary to provide sufficient chilling for high-chill cultivars, whereas protection against cold/frost damage might be needed for other cultivars. If cultivars will be evaluated in open field with no protection, it is better to only use cultivars adapted to the local environments to avoid damages from frost, heat, and other stresses. Additionally, large-scale genotype screening will also require phenotyping capacity to evaluate a wide range of shoot, root, and fruit-related traits, which is expensive and labor intensive.

To select drought-tolerant, high-yielding blueberry plants more efficiently, modern high-throughput plant phenotyping technologies involving multi-modal imagery, artificial intelligence (AI), sensing, and robotics will be needed. As drought influences the plant’s physiological, morphological, and physio-chemical properties, these changes can be rapidly characterized by using non-destructive imaging and sensing techniques. Ge et al. (2016) used red-green-blue (RGB) and visible near-infrared (VNIR) hyperspectral cameras to phenotype the drought tolerance of two maize genotypes. The results indicated that RGB images accurately predicted the shoot fresh weight, dry weight, and leaf area, whereas the leaf spectra accurately predicted leaf water content. Kim et al. (2020) used RGB, near-infrared (NIR), thermal and fluorescence imagery to quantify drought tolerance in the rice. Results indicated that these imaging modalities were successful in accurately measuring the plant area, water content, plant temperature, and photosynthetic efficiency, respectively. Kim et al. (2015) used short-wave infrared (SWIR) imagery to derive drought-sensitive indices for early detection of water stress. Their result indicated that images in the range of 1400 – 1450 nm are highly correlated with the leaf water content. Beyond-visual range imaging, including hyperspectral, multispectral, fluorescence, and thermal imaging together with artificial intelligence (AI) have also been successfully used as a high-throughput phenotyping modality in other major row crops including maize (Ge et al., 2016; Rehman et al., 2020; Ma et al., 2021; Rehman and Jin, 2022), soybean (Santana et al., 2022), and wheat (Correia et al., 2022). High-throughput phenotyping studies for blueberries have been relatively limited compared to other crops. Recent high-throughput phenotyping research for blueberries has investigated the morphological traits for identifying machine-harvestable genotypes (Patrick and Li, 2017). The study reported a strong correlation between traditional growth indices and quadcopter-based image-derived blueberry volume. The new drought tolerance studies on blueberries are still using the traditional low throughput techniques (Estrada et al., 2015; Balboa et al., 2020; Molnar et al., 2022; Zhang et al., 2022) and therefore need to be updated as has been adopted in other crops.

Traditional blueberry breeding relies mainly on phenotypic information for parent/offspring selection, which can take 15–20 years for cultivar release (Hancock et al., 2008). On the other hand, molecular-assisted breeding has a great potential to improve the efficiency of traditional breeding using molecular tools such as marker-assisted selection and genomic selection. Marker-assisted selection refers to the selection of parents or offspring based on a few markers closely linked to the genes or traits of interest (Xu and Crouch, 2008), which is recommended for qualitative traits controlled by a few genes and with a high heritability (Jannink et al., 2010). Genomic selection, which aims to predict the performance of an individual based on simultaneously estimated effects of all markers across the genome, is recommended for quantitative traits controlled by many and small-effect genes (Jannink et al., 2010). Both marker-assisted selection and genomic selection offers the opportunity to identify optimal individuals using marker information, potentially speeding up drought tolerance breeding by substituting challenging and costly phenotyping protocols.

The potential of molecular breeding to enhance drought tolerance is impeded by a lack of understanding on the genetic basis of drought tolerance in blueberries. The implementation of marker-assisted selection requires preidentified QTL or genes closely related to the trait of interest, often through QTL mapping or GWAS (Edger et al., 2022). In blueberries, QTL have been identified for traits related to fruit quality, machine harvesting, and climatic adaptation (Edger et al., 2022). Nevertheless, no QTL mapping or GWAS have been reported for drought tolerance, largely due to similar challenges encountered in large-scale cultivar evaluation for drought tolerance. The blueberry community does have rich genomic resources and tools for QTL/gene discovery, including high-quality reference genomes, next-generation genotyping platforms, linkage maps, and analysis software for polyploid species (Edger et al., 2022). If challenges related to large-scale drought tolerance evaluation can be addressed, QTL mapping or GWAS can be made possible by conducting drought evaluation on segregating mapping populations or diverse panels. As a result, a deeper understanding of the genetic architecture of drought tolerance will help researchers to identify optimal breeding strategies: marker-assisted selection if large-effect QTL or genes are discovered, or genomic selection if drought tolerance is highly quantitative in nature.

In addition to QTL mapping and GWAS, transcriptomics is another useful tool to advance gene discovery for drought tolerance in blueberries, especially when used alongside other QTL/gene mapping methods. Transcriptomics involves the study of transcriptome, which refers to the complete set of RNA transcripts or messenger RNA (mRNA) that are produced by the genome under specific conditions or in a specific cell (Vulimiri et al., 2014). Advances in next-generation RNA sequencing technologies have provided researchers with powerful tools to study gene expression patterns in greater detail, with higher accuracy, and at a more affordable price (Stark et al., 2019). As discussed in section 3, which focuses on blueberries’ molecular response to drought, gene expression analysis of targeted transcription factor and gene families has revealed their significant role in blueberries’ drought responses. For example, MYB TF family (Zhang et al., 2020), NAC TF family (Liang et al., 2019), and LEA family (Balboa et al., 2020) have all been implicated. Since drought tolerance involves complicated metabolic and signaling pathways, future studies should analyze transcriptome rather than single gene families to better reveal the molecular mechanism of drought tolerance. Additionally, transcriptome analysis has been combined with QTL mapping to facilitate candidate gene discovery such as for leaf development in alfalfa (Jiang et al., 2022), hypocotyl elongation in rapeseed (Luo et al., 2017), pericarp thickness in sweet corn (Wu et al., 2020), and heat-tolerance in tomato (Wen et al., 2019) to facilitate candidate gene discovery. Joint QTL mapping and transcriptome analysis can be potentially useful for dissecting the genetic architecture of drought tolerance in blueberries. While molecular information can be instrumental for cultivar development, it is essential to develop a systematic approach to improve drought tolerance while maintaining an ideal level of other traits such as yield, fruit quality, and disease tolerance.

Drought-tolerant blueberry cultivars must satisfy multiple criteria. At the minimum, they should demonstrate the resilience to drought stress and the capacity for rapid recovery from any damage incurred. Furthermore, it is essential for blueberry plants to maintain overall health, ensuring sustainable production over the long term. Exceptional cultivars go beyond these basic requirements, exhibiting the ability to uphold acceptable fruit quality and yield even under drought stress, all while preserving their long-term health and productivity.

To breed blueberries with ideal drought tolerance, comprehensive research efforts are essential across several key areas to effectively address challenges in blueberry drought research: (1) drought tolerance evaluation to include a broader range of traits, such as root architecture and fruit-related performance under drought stress, to establish stronger association between physiological and molecular signals with drought tolerance mechanisms; (2) large-scale drought tolerance screening across diverse blueberry germplasm to uncover various drought tolerance mechanisms and valuable genetic resources; (3) high-throughput phenotyping for drought-related traits to enhance the efficiency and affordability of drought phenotyping; (4) identification of genetic architecture of drought tolerance using various mapping technologies and transcriptome analysis; (5) tools to facilitate molecular-assisted breeding for drought tolerance, such as through marker-assisted selection and genomic selection; and (6)investigation of the interactions between drought and other stresses, such as heat and mineral soil conditions, to develop stress resilient genotypes.