Chronic NaCl Exposure Induced Significant Ionic Stress Tolerance in Arabidopsis thaliana as Indicated by Increased Growth Following an Acute Ionic Stress

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To accommodate increasing agricultural demands associated with increasing population, efforts are being made to uncover and improve native mechanisms plants use to cope with abiotic stresses. One type of abiotic stress, high soil salinity, has been shown to rapidly depolymerize microtubules. The cellulose synthase complex, which is responsible for production of cellulose in plants, relies on microtubules for functionality. The complex includes a recently discovered group of companion proteins that accelerate microtubule repolymerization following an ionic stress-induced degradation. Given their compensatory nature, we would expect that an upregulation of companion proteins would increase a plant’s tolerance to ionic stress. If regulated by ionic stress, we would also expect to observe an increase in companion protein expression while exposed to high saline conditions. Therefore, we hypothesized that chronic exposure to ionic stress will lead to increased tolerance for subsequent ionic stress. This study showed that prior exposure was sufficient to improve tolerance as shown by increased growth and microtubule repolymerization after an acute ionic stress, relative to plants without prior chronic ionic stress. Although not conclusively shown, this study supports upregulation of companion protein transcription in response to ionic stress.

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Introduction

It is not without justification to say that agriculture makes up a substantial component of the world’s economy (Holcomb et al. 2014). The biomass of plants provides the world with many essential products that are used daily. Plant biomass primarily consists of plant cell walls, which provide the major sustainable resource for many human products including feed, food, and fuel (Somerville et al. 2010). The primary mass contribution of plant cell walls is cellulose, which is also the most abundant biopolymer on Earth (Endler et al. 2015). Consisting of microfibrils formed by linked glucan chains and stabilized by intra- and inter- molecular hydrogen bonds, cellulose is essential for plant development, directed cell growth, and total plant structure, as the main source of cell wall tensile strength (Heredia et al. 1995; McFarlane et al. 2014).

Environmental conditions play a large role in determining numerous characteristics of plants, such as rate of development, size, and overall structure. Abiotic environmental stresses, such as salinity, drought, and cold, can result in severe yield losses for all major plant crop species (Endler et al. 2015). In fact, it is estimated that the yield loss due to abiotic stresses may be as large as 50% for various crop species (Boyer 1982). Products that reduce or eliminate such yield losses make up a large industry in itself, with US farmers spending over $29 billion on fertilizers and protectants annually (Holcomb et al. 2014).

Given that cellulose is such an integral component of the cell wall and plant biomass, an understanding of the cellular machinery that produces it is essential for improving tolerance to abiotic stresses. Cellulose is synthesized by a microtubule-guided cellulose synthase complex (CSC) embedded in the plasma membrane (Endler et al. 2015). The keystone of the CSC is the cellulose synthase (CesA) enzyme itself. Using Arabidopsis thaliana as a model, fluorescently labeled CesA proteins have been observed as motile foci, demonstrating that the enzymatic activity of CesA is guided by microtubules (Paredez et al. 2006). Depolymerization of microtubules has been shown to lead to the internalization of the CSC and inhibit cellulose production (Zhong et al. 2002). In a 2015 study by Endler et al., two proteins in the cellulose
synthase complex (CSC) of Arabidopsis thaliana were identified. Subsequently, these proteins, CC1 and CC2, have been termed companions of cellulose synthase proteins (CC). These CC’s exhibit numerous characteristics that warrant further investigation. Activity of both CC’s were shown to be induced primarily under saline conditions (Endler et al. 2015). Furthermore, CC’s were found to be unnecessary for
plant survival under non-saline conditions, but increase salt tolerance and survival under saline conditions (Endler et al. 2015). Redundancies were found amongst both companion proteins such that only one is required for adequate tolerance to acute NaCl stress (Endler et al. 2015). In case studies on cc1cc2, double mutants exposed to acute NaCl stress, adult plants contained significantly lower levels of cellulose, and, thus, a decreased rate of growth as compared to controls (Endler et al. 2015). Further research analyzed the mechanism by which companion proteins confer NaCl stress tolerance.

Exposure of plant cells to a NaCl solution induces both ionic and osmotic cellular stresses (Endler et al. 2015). However, it has been found that microtubules disassemble rapidly only after exposure to ionic stress (Komis et al. 2014). As noted previously, depolymerization of microtubules results in internalized CSC and ceases production of cellulose. As shown in Fig. 1, the companion proteins act to tether cellulose synthase to microtubule subunits. Upon depolymerization, CC’s promote microtubule reassembly. Enhanced microtubule reassembly enables faster recovery of cellulose synthesis during times of ionic stress.

FIG. 1 | Application of NaCl rapidly depolymerizes microtubules (Endler et al. 2015). The cellulose synthase complex (synthase (CesA) and companion proteins (CC)), requires microtubule organization for mobility and enzymatic activity (Heredia et al. 1995). CC’s help repolymerize microtubules following depolymerization due to ionic stress (Agrios 2005).

FIG. 1 | Application of NaCl rapidly depolymerizes microtubules (Endler et al. 2015). The cellulose synthase complex (synthase (CesA) and companion proteins (CC)), requires microtubule organization for mobility and enzymatic activity (Heredia et al. 1995). CC’s help repolymerize microtubules following depolymerization due to ionic stress (Agrios 2005).

While the role of companion proteins have been investigated in cases of acute NaCl stress, little is known about how long term exposure to NaCl stress affects their production and the total plant adaptation to ionic stress. Given sustained exposure to a stressor, organisms can adapt by upregulating production of proteins involved with tolerating the stressor, allowing it to survive exposure (Boyer 1982). Furthermore, in the event that adaptive tolerances are upregulated, the organism could withstand subsequent exposures at a higher level than was previously tolerable (Boyer 1982). This study investigated such a mechanism with regards to CC1 and CC2 in Arabidopsis thaliana.

As shown in Fig. 2, we hypothesized that chronic conditioning of Arabidopsis thaliana to 25mM NaCl would increase the rate of microtubule repolymerization and leaf area growth following a 4 hr 200mM NaCl acute stress test, as compared to those without chronic conditioning.

FIG. 2 | Chronic NaCl exposure suspected to induce upregulation of CC which will increase tolerance to ionic stress. Tolerance of conditioned plants were assessed by observing growth after exposure to an acute ionic stress. CC2 protein image generated used Phyre2 web portal for protein modeling (Schindelin, J. et al. 2012).

FIG. 2 | Chronic NaCl exposure suspected to induce upregulation of CC which will increase tolerance to ionic stress. Tolerance of conditioned plants were assessed by observing growth after exposure to an acute ionic stress. CC2 protein image generated used Phyre2 web portal for protein modeling (Schindelin, J. et al. 2012).

Methods

We grew Arabidopsis thaliana plants in a peat/vermiculite mix supplemented with Jack’s nutrient solution (~MiracleGro®). The plants were split into three conditioning groups: 25mM NaCl, 50mM sorbitol, and no conditioning (control). The NaCl group was grown with a modified version of Jack’s containing 25mM NaCl (concentration taken from Choi et al. 2013) for 14 days, which simulated a low level of ionic stress. The sorbitol group contained Jack’s with the addition of 50mM sorbitol. Since NaCl also induces osmotic stress, and we were only interested in how the plants adapt to long term ionic stress, the sorbitol was used to induce the equivalent of 25mM NaCl osmotic stress. This allowed us to attribute differences in growth or microtubule repolymerization in part to the CC’s involved in ionic stress rather than a physiological change associated with osmotic stress, like vascular adaptations. In order to simulate an acute ionic stress, we exposed all 3 groups (control/NaCl/sorbitol) to 200mM NaCl for 4 hours after the 2 week conditioning period (concentration and duration taken from Endler et al. 2015).

Growth Assay

Before and after the acute NaCl stress, we measured the average leaf area of the Arabidopsis plants. We imaged and measured leaf area in FIJI (Schindelin et al. 2012) as an indication of size and therefore growth, as shown by Weraduwage et al. 2015. Tukey-Kramer t-tests were used to compare mean leaf area by treatment, before and after acute stress test.

Root Integrity Assay

The roots were stained with calcofluor-white (CW) fluorescent dye, as described by Galbraith 1981. CW binds specifically to cellulose and not glucose or any of the other sugars comprising the cell wall(s)
making it an ideal stain for measuring cellulose. Calcofluor excitation occurs at 360 nm, while emission can be observed from 430-550 nm, which allowed us to separate its wavelength from the GFP-TUA6 on the microtubules. Cellulose was visualized with Zeiss Axiozoom, and average RFU/pixel measured in FIJI image analysis software (Schindelin et al. 2012). Independent t-tests were used to compare treatment groups before and after acute stress.

Microtubule Assay

We grew Arabidopsis thaliana with GFP-TUA6 marked microtubules (Gilroy lab, University of Wisconsin-Madison) in order to visualize the de- and re-polymerization of the microtubules after an acute NaCl stress. We used the Zeiss LSM510 confocal microscopy at the Newcomb Imaging Center, Department of Botany at the University of Wisconsin-Madison to observe the GFP labeled microtubules. GFP-TUA6 excitation occurs at 488 nm while emission can be observed from 285-365 nm. We observed the microtubules immediately before treatment and then every hour for 4 hours after the acute stress in order to visualize and compare the breakdown and repair of the microtubules between conditioning treatments.

Results

Average leaf areas between all conditioning treatments before stress were not significant (p= 0.87, 0.92, 0.99, respectively, Tukey-Kramer HSD). NaCl conditioned plants showed higher average leaf area following the acute stress test as compared to the control, however no significant difference was observed between NaCl-sorbitol or sorbitol-control (p=0.05, 0.12, 0.92, respectively, Tukey-Kramer HSD). Results are depicted in Fig. 4.

As depicted in Fig. 3, GFP-labeled microtubule images in the chronically conditioned plants showed faster microtubule reorganization and repolymerization upon exposure to acute NaCl stress than the control. Due to low sample size of images and inability to quantify repolymerization, statistical analysis was not performed. All results represent the average of the overall observed characteristics for each treatment group at a given time.

As shown in Fig. 5, the root integrity assay exhibited no statistical difference in the cellulose or structural composition of the roots before or after acute NaCl stress (t(20)= 2.03, p=0.25; t(20)= 2.03, p=0.87, two-tailed, respectively).

FIG. 4 | Mean leaf area prior to and four days following 200 mM salt stress in A. thaliana conditioned with 25 mM salt, 50 mM sorbitol, and no conditioning (p≤0.05, independent t-tests, error bars: ± 1 SE, n=30). Asterisks indicate significance.

FIG. 4 | Mean leaf area prior to and four days following 200 mM salt stress in A. thaliana conditioned with 25 mM salt, 50 mM sorbitol, and no conditioning (p≤0.05, independent t-tests, error bars: ± 1 SE, n=30). Asterisks indicate significance.

Discussion

Growth Assay

A significant increase in leaf area growth,
after an acute NaCl stress, was observed in plants exposed to chronic NaCl stress as compared to plants not exposed to chronic NaCl stress. This implies that prior conditioning with NaCl leads to increased ionic stress tolerance, which could be related to an increase in the expression of factors involved in recovering cellulose synthesis. Potential factors aiding in recovery, that could be upregulated by ionic stress, include the companion proteins described in Endler et al. 2015. However, analysis of transcription levels following ionic stress would need to be performed in order to directly support the regulatory mechanism outlined in Fig. 2.

FIG. 3 | Microtubule repolymerization in A. thaliana over time after acute 200mM NaCl stress test in salt and control conditioning groups. Scale bar represents 20μm. Confocal imaging was performed using the Zeiss LSM510 at the Newcomb Imaging Center, Department of Botany, UW-Madison.

FIG. 3 | Microtubule repolymerization in A. thaliana over time after acute 200mM NaCl stress test in salt and control conditioning groups. Scale bar represents 20μm. Confocal imaging was performed using the Zeiss LSM510 at the Newcomb Imaging Center, Department of Botany, UW-Madison.

Microtubule Repolymerization Assay

Due to the highly complex nature of microtubules, quantification of the results obtained from the microtubule repolymerization assay was not feasible. However, the images obtained qualitatively suggest that the group chronically conditioned with NaCl exhibited increased microtubule repolymerization and stabilization as compared to the control. This qualitative assessment is demonstrated by the greater density of microtubule organization seen in the time course images of the NaCl conditioned group.

Root Integrity Assay

Any damage to the roots of a plant would hinder its ability to absorb micronutrients and water, ultimately, affecting the overall growth of the plant. In order to control for any difference in growth due to root damage by chronic or acute NaCl exposure, we examined the health of the roots by microscopy using the cellulose stain calcofluor white in the root integrity assay. No significant difference in root health between treatment groups was observed, suggesting that the observed difference in leaf growth was not due to root damage.

FIG 5. a) Root integrity in A. thaliana assessed before and after acute NaCl stress by measuring calcofluor fluorescence with Zeiss Axiozoom. Box plots represent average fluorescence per pixel. Conditioned plant roots did not differ significantly from control before acute NaCl stress (t(20)= 2.02619, p=0.2504, two-tailed) and after (t(20)= 2.03452 p=0.8740, two-tailed). b) Sample examination ofroot health by calcofluor stain. Average fluorescence measured 1nun from root tip using FIJI image analysis software.

FIG 5. a) Root integrity in A. thaliana assessed before and after acute NaCl stress by measuring calcofluor fluorescence with Zeiss Axiozoom. Box plots represent average fluorescence per pixel. Conditioned plant roots did not differ significantly from control before acute NaCl stress (t(20)= 2.02619, p=0.2504, two-tailed) and after (t(20)= 2.03452 p=0.8740, two-tailed). b) Sample examination ofroot health by calcofluor stain. Average fluorescence measured 1nun from root tip using FIJI image analysis software.

Conclusions

These results allow us to support our hypothesis, suggesting that prior chronic NaCl stress does induce an adaptation to deal with acute ionic stress, as measured by leaf area and microtubule repolymerization.

Due to complexities inherent in the repolymerization of microtubules, analysis is currently restricted to qualitative observations. In order to more accurately quantify a change in the rate of repolymerization, a more thorough understanding of microtubule formation and degradation is needed. The ability to apply statistical analysis would allow for greater support of the ionic stress recovery mechanism. Direct quantification of CC protein production and accumulation would also be needed for elucidation of the recovery pathway.

The ability for plants to upregulate companion protein production represents an evolutionary advantage to environments in which fluctuations in salt stress are a possibility (Boyer et al., 1982; Wang et al., 2007). Companion proteins confer adaptive resistance to salt stresses and other ionic stresses, and would have likely evolved a mechanism for regulation in response to a sustained stress exposure. Studying the underlying mechanisms of salt stress adaptation could be useful for agricultural purposes. Overall, this study offers new insights into the regulatory properties of companion proteins of the CSC, and enables further research into manipulation of these regulatory properties.

Acknowledgements

We would like to thank Simon Gilroy and Sarah Newcomb of the Gilroy Laboratory, University of Wisconsin-Madison and Michelle Harris, Seth McGee, and Josh Pultorak from the Biology Core Curriculum Program, University of Wisconsin – Madison for their contributions in executing this research study.

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