Persistent water shortages and scarcity are major global challenges that affect the livelihoods of more than half of the world’s population, many of whom live in the least developed parts of the world (FAO 2020, UNESCO 2021). The prudent use of the increasingly limited freshwater resources available especially in water scarce regions is necessary to maintain food security and general quality of life. As agriculture is predominantly the largest withdrawer of global freshwater resources, accounting for close to 70% of all freshwater withdrawals, strategies that maximise agricultural water utility are paramount to reducing global water scarcity (Hoogeveen et al. 2015, Gleick and Cooley 2021). Reducing the water used for the irrigation of crop plants is one such promising strategy that could help optimise agricultural water use. This is because irrigation accounts for the bulk of the water used by modern agriculture and as such, even a marginal decrease in plant water use could result in a noticeable decline in agricultural freshwater consumption (de Avila et al. 2015, Cao et al. 2021).
Numerous strategies to improve the efficiency of freshwater use by plants have been developed with some showing great promise for enabling a more sustainable path for agricultural water use management. Of these interventions, one of the most interesting is through the genetic manipulation of stomatal density, which has been shown to improve both drought resistance and Water Use Efficiency (WUE) in plants through limiting transpiration, which is the main source of water loss from plants (Bertolino et al. 2019, Buckley et al. 2020). Reducing stomatal density by manipulating the expression of different members of the Epidermal Patterning Factor (EPF) family of genes has been demonstrated to confer improved drought resistance and water use efficiency. This has been shown to be the case in Arabidopsis (Franks et al. 2015, Hepworth et al. 2015, 2016), barley (Hughes et al. 2017), rice (Caine et al. 2019, Mohammed et al. 2019) and wheat (Dunn et al. 2019). The wide use of this strategy could potentially decrease the water footprint associated with plant productivity and thus have a great impact in dealing with the prevalent global water shortages.
Despite the beneficial traits of manipulating the EPF genes, little is known about how changes in stomatal density affect root architectural development with only a few papers like Mohammed et al. (2019) showing how changes in the EPF1 gene affect rice root aerenchyma. This deficit in studies looking at root properties is mainly due to the fact that roots are often obscured within opaque soil and thus are frequently neglected as they are not easily studied using traditional methods of plant analysis. This is even more apparent in the model plant, Arabidopsis, which is a relatively small plant whose root properties when grown in soil are seldom measured in literature. In many of the Arabidopsis experiments, roots are often grown in colourless growth media such as agar gel (French et al. 2009, Xiao et al. 2015), hydroponic solutions (Dayod et al. 2013, Strehmel et al. 2014) or even artificially synthesised transparent “soil” (Downie et al. 2012, Ma et al. 2019). Although these techniques provide useful insights into how specific gene alterations may affect root growth, they lack a specific dimension patterning to the actual performance of Arabidopsis roots in soil, their natural growth media, soil.
Studying Arabidopsis root properties in soil using the traditional method of root washing is difficult due to the fact that Arabidopsis roots are very thin (48–150µm thickness) and fragile, resulting in significant losses during the washing process. As an alternative, some researchers have resorted to using angled plastic rhizotrons with uniformly graded sand, encouraging the roots to grow at the glass sand interface for ease of visualisation (Chapman et al. 2011). Hepworth et al. (2016) used vermiculite containing rhizotrons covered with a glass microfiber paper that prevented direct contact between Arabidopsis roots and the growth media thus enabling easy separation and analysis of the roots. All these techniques however do not give a true representation of how Arabidopsis plants would grow in natural environments. To counter for the challenges faced by the traditional root-soil analysis methods, Lucas et al. (2011), Seignez et al. (2010), Tracy et al. (2010) and more recently Morris et al. (2017) used more advanced X-Ray CT scanning to reveal Arabidopsis roots in natural soil. Most of these studies, however, did not reveal the entirety of the root architecture of the Arabidopsis plant. In the case of Lucas et al. (2011) (scan resolution18 µm pixel− 1), the researchers resorted to using other non-soil based methods to quantify root architectural properties in the mutants they studied whilst Tracy et al. (2010) (scan resolution16µm pixel− 1) only as a proof of concept showed only a single grayscale image of an arabidopsis without attempting to segment out the roots in the image. Seignez et al. (2010) (scan resolution 10µm pixel− 1) on the other hand in a soil pollution study investigated only a small root section of Arabidopsis halleri (Similar genus to Arabidopsis Thaliana) in contact with contaminated soil as opposed to revealing the entire root architecture of the plants grown. Only Morris et al. (2017) (scan resolution not specified) in a review looking at how roots are shaped by different stimuli by goes into greater detail of arabidopsis root architecture. In this study they looked at a time series of growth of an Arabidopsis seedling growing in soil for a 21 day period. Even in this experiment however, less than half of the entire core was scanned to reveal root architecture, leaving out a significant portion of the growing roots unimaged.
In-spite of the challenges associated with studying Arabidopsis in mineral soil as outlined above, here we aim to use soil based systems to better understand the consequences of alterations in stomatal density via manipulation of the EPF family of genes on root properties in Arabidopsis. This study compares three different Arabidopsis lines with differing stomatal densities; the wild-type control (Col-0), epf2-1 (a mutant that has an increased stomatal density) and EPF2OE (a transgenic line that overexpresses EPF2 gene resulting in greatly reduced stomatal density). Our specific objectives were to (a) Estimate the WUE of the EPF mutants as compared to wild type plants when grown under controlled conditions in soil using biomass and Δ methods, (b) Reveal whole root architectural properties of the different Arabidopsis lines when grown in soil using high-resolution synchrotron imaging scanning as well as Neutron CT and (d) Compare how the roots of these different lines interact with the soil pores.