Phenotypic plasticity is an important property that allows plants to respond to a wide range of different environments. This feature is influenced by internal and external conditions that modify developmental processes. Arabidopsis thaliana
) is widely distributed around the northern hemisphere and consequently is subjected to diverse environmental conditions, generating different natural variants called accessions [1
]. The Arabidopsis
accessions are an important genetic resource to identify mechanisms underlying plant development and stress tolerance as plant genotypes are constantly shaped by biotic and abiotic factors [2
]. These phenotypic and genetic variations have enabled the characterization of responses in Arabidopsis
natural variants using a range of different approaches [3
Water deficit is an abiotic stress that affects plant development and productivity. Water availability can be altered by changes in solute concentration (i.e., sugars, salt, inorganic cations and anions) during drought, cold stress, and freezing [9
]. Some phenotypic and genetic analyses have identified tolerant accessions that can be useful to study the traits related to water deficit tolerance [11
] (for a review see [13
Although stress affects the whole organism [14
], leaves and roots display different responses in order to reduce water loss and promote water foraging for survival [18
]. Accordingly, plant roots act on the frontlines by sensing the water deficit, adjusting osmotic homeostasis, forcing water entrance, and avoiding water loss through the accumulation of compatible solutes inside the tissues [19
]. This rapid root response allows increased water uptake to maintain cellular turgor and reduce the negative effects in the leaves; however, when the stress becomes more severe, the growth in all tissues is highly compromised and it can cause the plant’s death [18
]. All these cues are later communicated to the shoot, which might respond with reduced growth rates, stomata closure, or rapid senescence, but the root is the main organ that reads and responds to the water availability status [19
]. Therefore, the root system not only represents a key model for studying water stress, but it is highly relevant to characterizing how the whole plant figures out strategies to face, tolerate, and recover from water stress. It is important to understand how these strategies have diverged in natural populations to deepen the genetic basis of the plant’s responses to drought stress.
root system is composed of primary and lateral roots with identical radial organization. In the primary root three distinct zones are distinguished by their abilities to proliferate, elongate, or differentiate [24
]. The proliferative zone is at the root apical meristem (RAM), which contains the stem cell niche (SCN) that is formed by four different sets of stem cells (also called initial cells) that yield all root cell types [27
]. These initial cells surround an organizer center called the quiescent center (QC) with very low mitotic activity and the capacity to produce short-range signals that are important for maintaining the initial cells in an undifferentiated state [28
]. Additionally, the RAM can be subdivided in two domains: the proliferation domain (PD) and the transition domain (TD). In the former, cells proliferate for 4–6 cycles and maintain a relatively small size, whereas in the TD, cells have a lower proliferation rate and they start to enlarge [24
]. The cells that stop proliferating and elongate anisotropically at very fast rates are confined to the elongation zone (EZ), whereas in the differentiation zone (DZ) cells acquire their final characteristics [25
]. Proliferation and differentiation are two interlinked processes in which the cells that are produced in the meristematic region are then displaced from it to the elongation zone towards the differentiation zone. It has been shown that both processes contribute to the final organ size [31
The imminent and drastic environmental changes caused by global warming and climate change have drawn global attention to understanding how plants cope with water deficit and osmotic stresses. Despite the vast literature dealing with the issue, little attention has been paid to the plant organ directly facing the stress on the frontline: the root. Therefore, in this study we decided to use nonionic solutes such as mannitol or sorbitol in order to evaluate root responses to water deficit, thereby changing the osmotic potential without adding ionic effects. We analyzed morphological alterations of root cells in response to hyperosmotic shock stress treatments in 15 Arabidopsis
accessions, some of them characterized as salt-tolerant accessions based on their responses in aerial tissues [12
]. We found that hyperosmotic stress inhibits root cell proliferation and elongation but does not interfere with QC identity or SCN morphology. Furthermore, under hyperosmotic stress, cortical and epidermal cells swelled and displayed a premature transition from the TD to the EZ and from the EZ to the DZ in all accessions. Interestingly, cell swelling occurred when the plant was subjected to a rapid osmotic shock treatment, with either an increase or decrease of solutes in the growth medium. The phenotypic primary root analysis revealed that root growth of the accessions Sg-2 and Ws was less affected by osmotic stress treatments than root growth of Cvi-0, which had an intermediate effect; whereas Col-0 and Ler
accessions were most severely affected. In addition, Sg-2 followed by Cvi-0 were shown to be the most resilient accessions in their recovery from strong hyperosmotic stress to control conditions. Unexpectedly, we did not find a correlation between the resiliency and the expression of different osmotic stress-related genes, suggesting that their increase in gene expression is not necessary to induce plant resilience.
2. Materials and Methods
The Arabidopsis thaliana accessions used in this work were: Büchen (Bch-4; ID: SJA26800), Buchschlag (Bu-5; ID:SJA02900), Burren (Bur-0; ID: SJA04400), Llagostera (Ll-1; ID: SJA33200), Schwieggershausen (Sh-0; ID: SJA21600), Sankt Georgen (Sg-2; ID: SJA21500), Wildbad (WI-0; ID: SJA25100), and Zurich (Zu-0; ID: SJA26400) from Riken Institute, Yokohama, Japan and Cape Verde Islands (Cvi-0; ID: N1096), Frankfurt (Fr-2; ID: N1168), HR (HR-5; N22205) and Tabor (Ta-0; ID: N1548) from Nottingham, England Arabidopsis Stock Centre (NASC). Columbia (Col-0), Landsberg erecta (Ler) and Wassilewskija (Ws) were accessions routinely used in our laboratory for more than 15 years.
2.1. Plant Growth Conditions
Seeds from different Arabidopsis accessions were disinfected with 20% sodium hypochlorite and 0.01% of Tween 20 for 15 min and stratified at 4 °C for 5 days under dark conditions, and sown on square Petri dishes containing MS medium (0.2 × Murashige and Skoog salts (MP Biomedicals; Irvine, CA, USA), 0.05% MES (Sigma-Aldrich; St. Louis, MO, USA), 1% sucrose (Sigma-Aldrich), and 1% agar (Becton, Dickinson and Company; Franklin Lakes, NJ, USA)), at pH = 5.6. For osmotic treatments, five days after sowing (5 dps) seedlings were transferred to MS medium (Control) or MS supplemented with concentrations of mannitol or sorbitol as indicated in each case and grown for 24 h. For the recovery assays, 5-dps seedlings were grown in MS medium with 400 mM of mannitol (changing the plants to a new medium with 400 mM of mannitol every week). Afterwards, the seedlings were returned to control conditions (MS medium) for 10 days to finally transfer them to soil for 7 days. For the hypoosmotic assay, 5-dps seedlings were transferred for one day to hyperosmotic stress conditions (300 mM mannitol) and then returned to control conditions for another day. In all cases plants were grown in a chamber at 22 °C under long-day (LD; 16 h light/8 h dark) conditions with a light intensity of 110 µm−2 s−1.
2.2. Osmotic Potential Measurement
To measure osmotic potential (ψπ), we used the vapor-pressure osmometer (VPO) Wescor, model VAPRO Model 5600 (ELITech group; Puteaux, France). The instrument has a small depression where a filter paper disk is filled with 10 L of the solution to measure (100 mM, 200 mM, and 300 mM of sorbitol and 100 mM, 200 mM, 300 mM, and 400 mM of mannitol).
2.3. Pseudo-Schiff Assay
For root cellular analysis, the roots of 5-dps seedlings grown in MS medium were transferred for one day to MS medium (control) or to MS supplemented with 300 mM of mannitol, and then were fixed and stained according to a modified Truernit protocol [33
]. This was done as follows: seedlings were fixed in a solution of 50% methanol and 10% acetic acid at room temperature (this can be done for 30 min up to two weeks). After fixation, roots were washed three times with distilled water and then incubated for 30 min in 1% periodic acid. After the periodic acid, plants were washed three times with distilled water and placed for 2 h in 0.18 M sodium bisulfite, 0.15 N hydrochloric acid, and 100 μg/mL propidium iodide at room temperature. Seedlings were washed again three times with distilled water and placed in Hoyer’s solution (80% chloral hydrate and 10% glycerol) for microscopy observation.
2.4. Microscopy Visualization
Root tissues were visualized using Nomarski optics under an Olympus BX60 microscope with a dry 40× objective and photographed with an Evolution MP COLOR camera of Media Cybernetics. Confocal images were acquired using the Olympus FV 1000 microscopy with an oil immersion 40× objective.
2.5. Kinematic Analysis
For all the quantitative cellular analysis, cell size and root domains and zones, were obtained using Fiji software [34
]. The data were analyzed as previously described [35
]. The growth rate and root length of each accession were obtained by marking the position of the root tip every 24 h on the back of the plate, the results of which were digitalized and measured using Fiji software. The cell size profile along the apical–basal axis of the root was obtained by measuring each cortex cell length along the cell file from the QC (cell 1) up to the fully mature zone (20 or more cells after the cortical cell nearest to the epidermal cell with the first hair root). The characterization of root domains and zones was done using a method based on double mobile linear regressions of cell length distributions along the root longitudinal axis, as described by multiple structural change algorithm (MSC) [36
2.6. RNA Extraction and Quantitative RT-PCR
Five-days-old Col-0, Cvi-0, and Sg-2 seedlings were transferred to control or 300 mM mannitol supplemented media for 8 h. Total RNA was extracted from the whole roots using the RNeasy Plant Mini Kit (QIAGEN; Venlo, The Netherlands). Concentration and integrity of the extracted RNA were tested using a NanoDrop 2000c spectrophotometer (Thermo Scientific; Waltham, MA, USA) and bleach agarose gel electrophoresis (Aranda et al., 2012). RNA was then reverse-transcribed into cDNA with SuperScript III First-Strand Synthesis SuperMix (Invitrogen; Carlsbad, CA, USA). RT-qPCR was performed with SYBR Select Master Mix (Thermo Scientific) using the ΔΔCt
(AT4G00660) and AT5G15710 were used as reference genes. The primer sequences used in this study are listed in Table S1
. Each experiment was performed with three biological replicates.
2.7. Geometric Morphometric Analysis
The seedlings were grown for five days under control conditions and then transferred to either control, 100, 200, or 300 mM of mannitol and plates were scanned at 800 dpi resolution. For geometric morphometric analysis we used the Shape Model Toolbox software [37
] implemented for roots as RootScape [38
]. The model was made out of 20 landmarks as follows: one landmark at the base of the root, one at the tip of the root, two at the locations of the first and last elongated lateral roots along the primary root, and two at the widest points of elongated lateral roots at each side of the primary root. A total 14 pseudo-landmarks were placed evenly spaced between the landmarks; all the landmarks built a polygon that captured the convex hull-shape of the root architecture. This allometric model considered the length of the primary root, the branching pattern, and the angle of the primary root. Using the landmark data, a geometric morphometric principal component analysis was done with Procrustes for rotation and translation to the centroids in order to align the shapes, but without size normalization. Here we only showed the three principal components (PCs) that capture 93.7% of the variation as an arbitrary cutoff.
Water, which is taken up by the root system, is the most limiting resource for plant growth. Under water deficit, the root acts as a sensory system integrating changes in water content to respond accordingly. The osmotic adjustment occurs in the roots before the leaves to enhance turgor pressure for continued root growth and absorption of water and nutrients [59
]. Therefore, we used the primary root of Arabidopsis
to understand how it responds to hyperosmotic stress conditions and its variation in different accessions. This organ enabled us to perform in vivo quantitative cellular analyses of different Arabidopsis
accessions to evaluate how cell proliferation and differentiation are affected individually under these conditions and how resilient they are once the stressful condition has been removed.
In our study, we exposed plants to an osmotic shock, changing drastically and immediately the osmotic pressure of the medium. In the 15 tested accessions root growth was impaired under our stress conditions, affecting both primary and lateral root growth, which is consistent with what has been previously reported for Col-0 [39
]. Although different types of stress affect both primary and lateral root length, the latter is hypersensitive to salt stress in comparison to growth of the primary root [50
]. According to Julkowska and collaborators, under salt stress conditions there are four root strategies to cope with salt stress using three different parameters: Primary Root (PR) growth, Lateral Root (LR) growth, and LR number. One of their strategies implied that the PR growth was more affected than LR growth under long-term exposure to ionic stress conditions [60
]. Our geometric morphometric principal component analysis showed that the primary root length was the outlier to understand the impact of osmotic stress induced by mannitol in root architecture in the five accessions tested here. It could be very interesting to study the interplay between PR and LR growth in our experimental system during different developmental stages as it has been reported that the effect on the ratio of LR growth vs. PR growth changes with different developmental stages as well as with different experimental procedures [50
In the primary root, the altered cellular patterns resulting from root growth under stressful conditions could be a useful experimental subject to approach to further unravel the role of different components on morphogenetic patterns in this organ. For example, under ionic stress conditions in Arabidopsis
and under osmotic or water stress in maize, rice, and hybrid poplar, the cell expansion and cell production rate are affected, thus altering primary root growth [60
]. The strength of the osmotic stress also affects each root domain differently, i.e., the meristematic cell number that is reduced in severe stress (−1.2 MPa), affecting the size of the Arabidopsis
primary root [41
]. Likewise, the cells at the TD are very sensitive to diverse environmental cues such as gravity, light, humidity, and various types of stress [24
]. In maize and soybean, the relative elongation rate under water deficit is unaffected at the apical zone near the meristem, but it is inhibited throughout the elongation zone [22
]. This occurs due to cell-specific structural changes and metabolic properties towards stress conditions in the different zones and domains along the longitudinal root axes [66
Under our experimental conditions, the reduction in primary root growth could be explained by the decrease in the meristem cell number of the RAM and the shorter lengths of the differentiated cells in plants subjected to hyperosmotic stress conditions. Analogous to the results presented here, under ionic stress conditions, root proliferation, and elongation explained smaller RAM and EZ in the root [31
]. In addition, RAM differentiates prematurely under water deficit in wheat root [70
] and various species growing under diminished water availability; it has been suggested that this is an adaptive plant response to cope with this stress condition [70
]. In our study we showed that the decrease in size of the RAM is related to a premature transit in two points: the proliferation domain to the transition domain, as the number of cells in the proliferation domain changes under osmotic stress treatments as compared to our control treatment; and the transition domain to the elongation zone, as the cell size in the former is shorter in almost all the accessions tested.
Additionally, we found that some root cells (cortex and epidermis) at the intersection between the RAM and the EZ showed a radially swollen phenotype when plants are exposed to a hyperosmotic shock condition. A similar phenotype was reported in wheat, maize, soybean, rice, and Brachypodium
in response to osmotic and water stress conditions [31
]. In wheat, swollen cells stop proliferating as they no longer stain with tetrazolium violet, and also increase their proline content, indicating an osmotic adjustment [70
]. Furthermore, a drought-tolerant wheat cultivar has a lower percentage of swollen roots than another more sensitive cultivar [70
]. In contrast to wheat cultivars, in Arabidopsis
we found the same swollen cells in both sensitive and tolerant accessions. Moreover, our results indicate that this phenotype depends on the sudden osmotic potential change, which seems to be sparked by a lower (hypo) or higher (hyper) solute concentration shock on the medium, rather than an adaptive response to hyperosmotic conditions. Consistent with this observation, this swollen cell phenotype did not appear when plants were germinated and grown in 300 mM of mannitol, as these plants never experienced an osmotic shock.
Interestingly, this radial cellular expansion has been reported at the root apex of Arabidopsis
under salt stress [72
], in the lateral roots of plants under drought conditions [17
], in plants with either altered cell wall biogenesis [74
] or microtubule cytoskeletons [75
], or multivesicular body biogenesis [76
]. Given that swelling is determined by the physicochemical properties of the cell wall [77
], its occurrence is not surprising in the transition domain cells that are located between the RAM and the EZ in wheat [70
]. In Arabidopsis
, the transition domain has unique physiological properties such as alterations in their cell wall structure and vacuolization that enables fast length growth in the EZ [24
]. In contrast, cell morphology of the root SCN was unaffected by osmotic stress and it has been reported that the maintenance of functional SCN is an ability of the roots to withstand the concurrent environmental conditions, which allows roots to restore their growth dynamics when conditions are more favorable [78
According to our results, root cell proliferation is resilient, and once the hyperosmotic stress is withdrawn, normal growth partially recovers after one day, even if plants have been growing under highly stressful conditions such as 300 mM of mannitol. Although the root growth of all accessions tested here was affected, each accession had a different sensitivity, Sg-2, Ws, and Cvi-0 being less affected than Col-0 and Ler
. The recovery of root growth after strong osmotic stress (400 mM for 18 days) was also variable, since Sg-2 and Cvi-0 exhibited more tolerance than the rest of the accessions. Puzzlingly, these responses contrast to what has been previously reported for salt stress in aerial tissues, where Cvi-0, Sg-2, Col-0, and Ler
were the most sensitive accessions [12
]. Although we still do not know if these differences are due to the type of stress, we cannot rule out the possibility that the root and aerial tissues respond differently to osmotic stress. However, our results suggest that integrative studies that consider both the shoot and the root are crucial to define stress sensitivity. On the other hand, the resilience of Sg-2 was surprising, since its location of origin (Sankt Georgen, Germany) is a cool and wet climate, and yet it has been reported as a salt stress-sensitive accession [12
] raising questions about the adaptive nature of its phenotype. In contrast, Cvi-0 is native to a warm and dry climate [81
] and more resistant to various types of stress [82
], which might be expected since Cvi-0 is believed to be the result of a relatively recent introduction into the African continent, reflecting evolutionary bottlenecks, drift, and adaptive evolution.
The specific mechanism that Cvi-0 and Sg-2 use to resist the strong osmotic stress is still unknown. Although both accessions are able to induce some osmotic stress genes, the induction levels were similar or even lower than the ones we obtained with Col-0, a sensitive accession; therefore, other mechanisms could be involved. It has been reported that Cvi-0 has higher ABA levels than Col-0 [84
], which is related to the higher levels of RD29B
, a gene mainly controlled by ABA, observed in Cvi-0 as compared to Col-0, under control conditions. RD29A
decreased its expression in Cvi-0, but it is mainly induced through the ABA-independent pathway [86
]. The increase of RD29B
observed in Sg-2 could also be related to high levels of ABA. Therefore, ABA content or a constitutive activation of stress responses may be responsible for generating different sensitivities to osmotic stress, rather than changes in expression in some genes.
This work has uncovered quantitative cellular data that helps to explain how root development is affected by osmotic stress conditions, the natural variation on the plasticity of these mechanisms, and how these organ responses relate to tissue growth dynamics, cell proliferation, and differentiation. It has also been shown that this experimental system of altered growth and cellular dynamics may be a useful system to test the models of coupled cell proliferation and physicochemical properties in order to understand the emergence of cellular patterns and behaviors in complex organs, such as the Arabidopsis
]. Future experimental and “in silico
” approaches will be necessary to further unravel the developmental and genetic nature of stress responses and their evolution.