Abscisic Acid Inhibits Cortical Microtubules Reorganization and Enhances Ultraviolet-B Tolerance in Arabidopsis thaliana

Ultraviolet-B (UV-B) radiation is one of the important environmental factors limiting plant growth. Both abscisic acid (ABA) and microtubules have been previously reported to be involved in plant response to UV-B. However, whether there is a potential link between ABA and microtubules and the consequent signal transduction mechanism underlying plant response to UV-B radiation remains largely unclear. Here, by using sad2-2 mutant plants (sensitive to ABA and drought) and exogenous application of ABA, we saw that ABA strengthens the adaptive response to UV-B stress in Arabidopsis thaliana (A. thaliana). The abnormal swelling root tips of ABA-deficient aba3 mutants demonstrated that ABA deficiency aggravated the growth retardation imposed by UV-B radiation. In addition, the cortical microtubule arrays of the transition zones of the roots were examined in the aba3 and sad2-2 mutants with or without UV-B radiation. The observation revealed that UV-B remodels cortical microtubules, and high endogenous ABA can stabilize the microtubules and reduce their UV-B-induced reorganization. To further confirm the role of ABA on microtubule arrays, root growth and cortical microtubules were evaluated after exogenous ABA, taxol, and oryzalin feeding. The results suggested that ABA can promote root elongation by stabilizing the transverse cortical microtubules under UV-B stress conditions. We thus uncovered an important role of ABA, which bridges UV-B and plants’ adaptive response by remodeling the rearrangement of the cortical microtubules.


Introduction
Climate change and the frequent activities of humans have intensified the depletion of the ozone layer in the atmosphere, resulting in enhanced ultraviolet radiation reaching the Earth's surface. Plant growth and development are inevitably affected by ultraviolet-B (UV-B) radiation (280-320 nm), which has prime damaging effects among ultraviolet radiations of 200-400 wavelengths and has been a growing concern for years. Enhanced UV-B has severely harmful effects on plant growth and development by prompting DNA damage, membrane changes, and protein crosslinking [1][2][3], causing changes in vital physiology processes, such as decreased photosynthetic rate [4] and plant morphology along with hormonal and signal shifts [5][6][7]. However, the underlying physiological and molecular mechanisms in plants remain unclear.
As a crucial organ for sessile plants, the root system response to UV-B radiation is reported widely. UV-B irradiation has been shown to retard the growth of barley roots and induce subapical root swelling, which leads to cortical cell expansion and subsequent swelling of the lower part of the elongation zone [8]. The A. thaliana mutants rus1 and rus2,

Plants and Growth Conditions
The A. thaliana Columbia (Col-0) ecotype was used as the wild type for all experiments in this study. The Arabidopsis thaliana mutant aba3 (a mutant deficient in ABA synthesis) [18] and the mutant sad2-2 (sensitive to ABA and drought and tolerant to UV-B) [19,20] were used. The seeds were sterilized and incubated at 4 • C in the dark for 2 days in a Murashige & Skoog (MS) basal medium containing 1.5% sucrose (w/v) and 0.7% agar (w/v) and were placed in the growth cabinet under a 16:8 h light/dark cycle at 22 • C for 5 days and then harvested for experiments.

UV-B and Pharmaceutical Treatments
Healthy seedlings were irradiated with 90 mJ cm −2 of UV-B for 30 min. A spectral irradiance of 90 mJ cm −2 was determined with an Ultraviolet Crosslinker (midrange, 302 nm; UVP Co., Upland, CA, USA). The 30 min irradiation duration was selected because it was the shortest UV-B exposure period out of the 0 to 60 min monitored periods, which affected significant phenotypes ( Figure A1). After radiation, the seedlings were positioned upside down for phenotype analysis in six days, and their cortical microtubules were observed 1, 2, and 6 h post-radiation.

ABA Extraction and Quantification
Healthy seedlings that did or did not receive UV-B treatment were collected and ground in a cold extraction buffer containing 80% methanol and 1 mmol of L −1 butylated hydroxytoluene. After 4 h of shaking in the dark at 4 • C, the homogenates were centrifuged (8400× g, 20 min, 4 • C), and the pellets were re-extracted for 60 min in 1 mL of the same extraction solvent. The supernatants were transferred to a fresh glass tube and vacuumdried using Power Dry LL3000 (Heto-Holten, Allerød, Denmark). Extracts were dissolved and diluted in PBS buffer containing 1 mL L −1 Tween-20 and 1 g L −1 gelatin. ABA contents were determined using a Plant Hormone ABA ELISA kit (R&D, Emeryville, CA, USA) according to the manufacturer's instructions.

Cortical Microtubules Immunolabeling and Observations
Seedling roots exposed or not to the above-described treatment were fixed with 4% (w/v) paraformaldehyde to investigate the effects of UV-B radiation and ABA on the cortical microtubule array. A mouse monoclonal antibody against β-tubulin (Sigma, St. Louis, MO, USA) at a 1:300 dilution and an Alexa Fluor ® 488-conjugated goat antibody against rabbit IgG (Molecular Probes, Sigma, St. Louis, MO, USA) at a 1:200 dilution were used as the primary and secondary antibodies, respectively. Immunofluorescence images were collected using a Zeiss LSM 510 META confocal microscope (Analytik Jena AG, Jena, Germany). The samples were excited at 488 nm with a krypton-argon laser, and the emission from the Alexa 488 ® fluorochrome was detected using a bandpass filter ranging from 505-530 nm.

Phenotype Analysis
Six-day-old seedlings grown in MS medium with or without ABA treatment were positioned upside down after UV-B radiation and grown for 6 days. The phenotypes of treated seedlings were photographed, and the images of their root tips were enlarged using a Zeiss LSM 510 META confocal microscope at the indicated time. UV-B radiation caused leaf chlorosis and root retardation. The phenotypes were analyzed to investigate whether the swollen root tips were regulated by the cortical microtubule orientation/dynamics. Each experimental group contained at least 20 seedlings, and each experiment was repeated at least three times.

The Morphology of Root Cells Staining by PI and Observations
Healthy six-day-old seedlings of A. thaliana Col-0 and mutants sad2-2 and aba3 were arranged in MS medium containing 0.5 µm L −1 ABA for 2 days or imposed by UV-B radiation for 30 min following 1-day normal illumination. Then the root tips were stained with 5 µg mL −1 propidium iodide (PI) according to the kit instructions and photographed for morphology observations of the root epidermal cells. The morphology of root cells was visualized to investigate the effects of UV-B radiation on root cells. Each experimental group contained at least 20 seedlings, and each experiment was repeated at least three times.

Statistical Analysis
All values are expressed as the mean ± standard error (SE) (n ≥ 10) for each trait. The crooked root lengths and the diameters of the root transition zone of seedlings were measured by the software Image J. All variations between treatments were evaluated with the least significant difference (LSD) multiple range tests (p ≤ 0.05) using IBM SPSS Statistics 27 software.

ABA Strengthens the Adaptive Response to UV-B Stress in A. thaliana
To gain insights into the function of ABA in plants' response to UV-B stress, the effects of endogenous and exogenous ABA on A. thaliana were investigated. Plants of Col-0 and sad2-2 were used for the study. Col-0 seedlings irradiated with UV-B (90 mJ cm −2 for 30 min, which is determined by the pre-experiment ( Figure A1) without ABA feeding, displayed remarkable chlorosis and root growth inhibition and subsequently died at 6 DPR (days post-radiation). In contrast, those UV-B-irradiated plants treated with exogenous ABA remained green and mostly survived at 6 DPR ( Figure 1a). Moreover, the sad2-2 mutant survived longer with less chlorosis phenotypic characteristics than Col-0 seedlings under UV-B-irradiation, similar to the ABA-treated Col-0 seedlings ( Figure 1b). We, therefore, evaluated the endogenous ABA levels of Col-0 and sad2-2 under normal and UV-B-radiated conditions. UV-B induced ABA accumulation in both of the plants. Moreover, we noticed that the ABA concentrations of sad2-2 plants were 19.2 and 46.08 ng g −1 FW before and after UV-B radiation, respectively, which were much higher than that of the wild type (5.76 and 22.08 ng g −1 FW, respectively) as shown in Figure 1c. Because the sad2-2 plants were more tolerant to UV-B than the Col-0 (Figure 1a,b), we proposed that the ABA is positively involved in the plant's response to UV-B radiation.
All values are expressed as the mean ± standard error (SE) (n ≥ 10) for each trait. The crooked root lengths and the diameters of the root transition zone of seedlings were measured by the software Image J. All variations between treatments were evaluated with the least significant difference (LSD) multiple range tests (p ≤ 0.05) using IBM SPSS Statistics 27 software.

ABA Strengthens the Adaptive Response to UV-B Stress in A. thaliana
To gain insights into the function of ABA in plants' response to UV-B stress, the effects of endogenous and exogenous ABA on A. thaliana were investigated. Plants of Col-0 and sad2-2 were used for the study. Col-0 seedlings irradiated with UV-B (90 mJ cm −2 for 30 min, which is determined by the pre-experiment ( Figure A1) without ABA feeding, displayed remarkable chlorosis and root growth inhibition and subsequently died at 6 DPR (days post-radiation). In contrast, those UV-B-irradiated plants treated with exogenous ABA remained green and mostly survived at 6 DPR ( Figure 1a). Moreover, the sad2-2 mutant survived longer with less chlorosis phenotypic characteristics than Col-0 seedlings under UV-B-irradiation, similar to the ABA-treated Col-0 seedlings ( Figure 1b). We, therefore, evaluated the endogenous ABA levels of Col-0 and sad2-2 under normal and UV-B-radiated conditions. UV-B induced ABA accumulation in both of the plants. Moreover, we noticed that the ABA concentrations of sad2-2 plants were 19.2 and 46.08 ng g −1 FW before and after UV-B radiation, respectively, which were much higher than that of the wild type (5.76 and 22.08 ng g −1 FW, respectively) as shown in Figure 1c. Because the sad2-2 plants were more tolerant to UV-B than the Col-0 ( Figure 1a,b), we proposed that the ABA is positively involved in the plant's response to UV-B radiation. (c) Endogenous ABA levels of six-day-old seedlings from wild type and sad2-2 mutants were assayed using a Plant Hormone ABA ELISA kit 60 min after radiation began and 30 min after it ended. Each experiment was repeated at least three times. All the statistical analyses were conducted using the IBM SPSS Statistics 27 software. All values are expressed as the mean ± SE (n ≥ 20). Letters of a-c represent statistically significant groupings of p ≤ 0.05 based on a one-way ANOVA with the LSD multiple range tests.

UV-B Intervenes Root Growth through ABA Signaling
To further uncover if ABA accumulation strengthens the tolerance of plants to UV-B radiation, we used sad2-2 (high levels of endogenous ABA shown in Figure 1c) and aba3 (deficient in ABA synthesis) mutants. We analyzed their response to UV-B radiation at Figure 1. UV-B radiation increased endogenous ABA concentration in A. thaliana seedlings, and exogenous ABA supplementation protected seedlings from UV-B radiation. (a) Six-day-old wild-type (Col-0) seedlings vertically grown in MS medium with or without 0.5 µmol L −1 ABA were irradiated with 90 mJ cm −2 of UV-B light for 30 min and positioned upside down. Phenotypes of the seedlings were photographed 6 days post-radiation; (b) Six-day-old wild type and sad2-2 mutant seedlings vertically grown in MS medium were irradiated with 90 mJ cm −2 of UV-B light for 30 min and positioned upside down; (c) Endogenous ABA levels of six-day-old seedlings from wild type and sad2-2 mutants were assayed using a Plant Hormone ABA ELISA kit 60 min after radiation began and 30 min after it ended. Each experiment was repeated at least three times. All the statistical analyses were conducted using the IBM SPSS Statistics 27 software. All values are expressed as the mean ± SE (n ≥ 20). Letters of a-c represent statistically significant groupings of p ≤ 0.05 based on a one-way ANOVA with the LSD multiple range tests.

UV-B Intervenes Root Growth through ABA Signaling
To further uncover if ABA accumulation strengthens the tolerance of plants to UV-B radiation, we used sad2-2 (high levels of endogenous ABA shown in Figure 1c) and aba3 (deficient in ABA synthesis) mutants. We analyzed their response to UV-B radiation at the early stages. After exposure to UV-B radiation for 30 min, all the roots of the irradiated seedlings immediately stopped elongating (Figure 2a,b), and at 48 h, the cells of the lower parts of the elongation zone became visibly swollen ( Figure 2a). Moreover, compared with the weak response of sad2-2 and Col-0, aba3 was hypersensitive to UV-B. The root tips of the aba3 seedlings expanded into a nodule at 48hr DPR, which was larger than that of sad2-2 and Col-0. Hence, ABA-deficient mutants aba3 are not only generally stunted but also more sensitive than sad2-2 and Col-0 in response to UV-B stress. PI staining data showed that all root tissue of UV-B radiated seedlings at 1 DPR was seriously damaged, and the root epidermal cells of the aba3 mutant were significantly enlarged ( Figure 2c). These findings suggested that ABA deficiency aggravated the growth retardation imposed by UV-B radiation and influenced root morphology by inhibiting elongation and promoting the isotropic expansion of root tips.
Genes 2023, 14, 892 5 of 18 the early stages. After exposure to UV-B radiation for 30 min, all the roots of the irradiated seedlings immediately stopped elongating (Figure 2a,b), and at 48 h, the cells of the lower parts of the elongation zone became visibly swollen ( Figure 2a). Moreover, compared with the weak response of sad2-2 and Col-0, aba3 was hypersensitive to UV-B. The root tips of the aba3 seedlings expanded into a nodule at 48hr DPR, which was larger than that of sad2-2 and Col-0. Hence, ABA-deficient mutants aba3 are not only generally stunted but also more sensitive than sad2-2 and Col-0 in response to UV-B stress. PI staining data showed that all root tissue of UV-B radiated seedlings at 1 DPR was seriously damaged, and the root epidermal cells of the aba3 mutant were significantly enlarged ( Figure 2c). These findings suggested that ABA deficiency aggravated the growth retardation imposed by UV-B radiation and influenced root morphology by inhibiting elongation and promoting the isotropic expansion of root tips. The crooked root lengths of seedlings were measured, and those not exposed to UV-B radiation were used as controls (CK). All the statistical analyses were conducted using the IBM SPSS Statistics 27 software. All values are expressed as the mean ± SE (n ≥ 20). And letters of a-j represent statistically significant groupings of p ≤ 0.05 based on a one-way ANOVA with the LSD multiple range tests. (c) The images of the root epidermal cells staining by Propidium Iodide (PI) in three genotype seedlings. Healthy six-day-old seedlings were arranged in MS medium containing 0.5 μmol L −1 ABA for two days or imposed by UV-B radiation for 30 min following one day of normal illumination. Then the root tips were stained with 5 μg ml −1 PI and photographed to show that ABA deficiency aggravated the root growth retardation and influenced root morphology. Scale bars represent 30 μm.

UV-B Remodels Microtubule Arrangement in Root Tip
Microtubule dynamics are essential for root tip development. Therefore cortical arrays in the elongation zone of root tips were observed at different time points after UV-B stress. As shown in Figure 3, the intact microtubule network of the cell was severely disturbed in Col-0 after UV-B irradiation. Transversely oriented microtubules were completely disordered at 1 HPR, were transformed into short, curved punctate structures, and microtubule abundance decreased sharply. At 2HPR, the microtubule abundance began to increase, and microtubule arrays reappeared obliquely. At 6 HPR, a highly ordered cortical microtubule rearrangement was found to be arranged longitudinally parallel to the longitude axis. The crooked root lengths of seedlings were measured, and those not exposed to UV-B radiation were used as controls (CK). All the statistical analyses were conducted using the IBM SPSS Statistics 27 software. All values are expressed as the mean ± SE (n ≥ 20). And letters of a-j represent statistically significant groupings of p ≤ 0.05 based on a one-way ANOVA with the LSD multiple range tests. (c) The images of the root epidermal cells staining by Propidium Iodide (PI) in three genotype seedlings. Healthy six-day-old seedlings were arranged in MS medium containing 0.5 µmol L −1 ABA for two days or imposed by UV-B radiation for 30 min following one day of normal illumination. Then the root tips were stained with 5 µg mL −1 PI and photographed to show that ABA deficiency aggravated the root growth retardation and influenced root morphology. Scale bars represent 30 µm.

UV-B Remodels Microtubule Arrangement in Root Tip
Microtubule dynamics are essential for root tip development. Therefore cortical arrays in the elongation zone of root tips were observed at different time points after UV-B stress. As shown in Figure 3, the intact microtubule network of the cell was severely disturbed in Col-0 after UV-B irradiation. Transversely oriented microtubules were completely disordered at 1 HPR, were transformed into short, curved punctate structures, and microtubule abundance decreased sharply. At 2HPR, the microtubule abundance began to increase, and microtubule arrays reappeared obliquely. At 6 HPR, a highly ordered cortical microtubule rearrangement was found to be arranged longitudinally parallel to the longitude axis.

ABA Stabilizes the Microtubule Array and Reduces UV-B-Induced Reorganization
Given that UV-B irradiation resulted in the modulation of both ABA and c microtubules in the root, we next examined whether or not there is a potential link be ABA level and microtubule dynamics. The microtubule arrays of the transition zone roots were then monitored in the aba3 and sad2-2 mutants with or without UV-B rad Under normal conditions, the microtubule arrays in the aba3 mutant were few and tered compared to Col-0, whereas in sad2-2, they were thicker than the wild type. U UV-B radiation conditions, the cortical array in aba3 displayed an oblique and lengt arrangement which was more severe than the arrangements in Col-0, while in con the influence of UV-B on the cortical array in sad2-2 was relatively slight expressing a no lengthwise microtubule was observed ( Figure 4). The results revealed that tog with ABA, UV-B remodels microtubule dynamics of the root tip, which is support the fact that high endogenous ABA levels can stabilize the microtubules and reduc UV-B-radiation-induced reorganization.
We also evaluated the effect of exogenous ABA on the cortical arrays in Col-0 and sad2-2. As shown in the bottom row of Figure 4, the fluorescence intensity in the genotypes was higher than in untreated controls. More transverse cortical arrays observed in aba3 after applying exogenous ABA. Meanwhile, ABA-treated sad2-2 exh relatively thicker and denser, even bunched microtubules, as shown by the arrow others due to the higher endogenous ABA content. These results suggested that incr levels of ABA promote the stabilization of transverse cortical microtubules and re UV-B stress. Cortical microtubules in root cells of seedlings that did not undergo UV-B radiation were used as controls (CK). The yellow lines processed by software Image J represent the change of length, direction, and structure of the microtubules in the selected cells. Immunofluorescence images were collected using a Zeiss LSM 510 META confocal microscope. Each experimental group had at least 10 seedlings, and each experiment was repeated three times. Scale bars represent 10 µm.

ABA Stabilizes the Microtubule Array and Reduces UV-B-Induced Reorganization
Given that UV-B irradiation resulted in the modulation of both ABA and cortical microtubules in the root, we next examined whether or not there is a potential link between ABA level and microtubule dynamics. The microtubule arrays of the transition zone of the roots were then monitored in the aba3 and sad2-2 mutants with or without UV-B radiation. Under normal conditions, the microtubule arrays in the aba3 mutant were few and scattered compared to Col-0, whereas in sad2-2, they were thicker than the wild type. Under UV-B radiation conditions, the cortical array in aba3 displayed an oblique and lengthwise arrangement which was more severe than the arrangements in Col-0, while in contrast, the influence of UV-B on the cortical array in sad2-2 was relatively slight expressing as that no lengthwise microtubule was observed (Figure 4). The results revealed that together with ABA, UV-B remodels microtubule dynamics of the root tip, which is supported by the fact that high endogenous ABA levels can stabilize the microtubules and reduce their UV-B-radiation-induced reorganization.
We also evaluated the effect of exogenous ABA on the cortical arrays in Col-0, aba3, and sad2-2. As shown in the bottom row of Figure 4, the fluorescence intensity in the three genotypes was higher than in untreated controls. More transverse cortical arrays were observed in aba3 after applying exogenous ABA. Meanwhile, ABA-treated sad2-2 exhibited relatively thicker and denser, even bunched microtubules, as shown by the arrow, than others due to the higher endogenous ABA content. These results suggested that increased levels of ABA promote the stabilization of transverse cortical microtubules and reduces UV-B stress.  , sad2-2, and aba3 mutants, three genotypes. Cortical microtubules wer observed 1 h post-radiation by immunofluorescence microscopy in the root epidermal cells of seed lings treated as described above. The yellow lines processed by software Image J represent the chang of length, direction, and structure of the microtubules in the selected cells, and the yellow arrows in dicate bundles of the microtubules. Immunofluorescence images were collected using a Zeiss LSM 510 META confocal microscope. Each experiment was repeated three times. Scale bars in the fluo rescence images of microtubules represent 10 μm.

ABA Regulates Primary Root Tip Growth via Stabilizing Cortical Microtubule Arrays
To further explore whether root growth is regulated by ABA and microtubule skele ton, we analyzed primary root morphological characteristics and cortical microtubules o wild-type seedlings after exogenous ABA, taxol, and oryzalin feeding. Taxol is a microtu bule stabilizing agent, while oryzalin is a plant dinitroaniline herbicide and a specific de polymerization agent for plant microtubule skeletons. Both ABA-and taxol-treated plant displayed thicker and denser cortical arrays and longer and more root hairs than the con trol plants. The cortical arrays of 0.25 μmol L −1 ABA-and taxol-treated plants were quite similar ( Figure 5). In contrast, the primary root tips of the oryzalin-treated seedlings exhib ited a disordered cortical microtubules, resulting in growth inhibition, abnormal roo hairs, and apical expansion similar to that of UV-B irradiated seedlings (Figures 2a and  5). These data proved that a low level of ABA promoted primary root elongation and roo hair growth by stabilizing the transverse cortical microtubules. . UV-B radiation altered the cortical microtubules in root epidermal cells from seedlings of A. thaliana wild type (Col-0), sad2-2, and aba3 mutants, three genotypes. Cortical microtubules were observed 1 h post-radiation by immunofluorescence microscopy in the root epidermal cells of seedlings treated as described above. The yellow lines processed by software Image J represent the change of length, direction, and structure of the microtubules in the selected cells, and the yellow arrows indicate bundles of the microtubules. Immunofluorescence images were collected using a Zeiss LSM 510 META confocal microscope. Each experiment was repeated three times. Scale bars in the fluorescence images of microtubules represent 10 µm.

ABA Regulates Primary Root Tip Growth via Stabilizing Cortical Microtubule Arrays
To further explore whether root growth is regulated by ABA and microtubule skeleton, we analyzed primary root morphological characteristics and cortical microtubules of wild-type seedlings after exogenous ABA, taxol, and oryzalin feeding. Taxol is a microtubule stabilizing agent, while oryzalin is a plant dinitroaniline herbicide and a specific depolymerization agent for plant microtubule skeletons. Both ABA-and taxol-treated plants displayed thicker and denser cortical arrays and longer and more root hairs than the control plants. The cortical arrays of 0.25 µmol L −1 ABA-and taxol-treated plants were quite similar ( Figure 5). In contrast, the primary root of the oryzalin-treated seedlings exhibited a disordered cortical microtubules, resulting in growth inhibition, abnormal root hairs, and apical expansion similar to that of UV-B irradiated seedlings (Figures 2a and 5). These data proved that a low level of ABA promoted primary root elongation and root hair growth by stabilizing the transverse cortical microtubules.

ABA Reduces UV-B-Induced Root Tip Expansion
To better understand the root morphology response to ABA and UV-B radiation, we compared the diameters of the root transition zone of seedlings exposed to taxol, oryzalin, ABA, and UV-B. Statistical analysis revealed that the root elongation inhibition (Figure 2b) and apical expansion caused by UV-B was significantly weakened by ABA application, while there was no significant difference between ABA concentrations of 0.25 μmol L −1 and 0.5 μmol L −1 (Figure 6). Compared with the treatment of UV-B and oryzalin, low concentrations of ABA can reduce UV-B-induced root tip swelling, similar to taxol.

ABA Reduces UV-B-Induced Root Tip Expansion
To better understand the root morphology response to ABA and UV-B radiation, we compared the diameters of the root transition zone of seedlings exposed to taxol, oryzalin, ABA, and UV-B. Statistical analysis revealed that the root elongation inhibition (Figure 2b) and apical expansion caused by UV-B was significantly weakened by ABA application, while there was no significant difference between ABA concentrations of 0.25 µmol L −1 and 0.5 µmol L −1 (Figure 6). Compared with the treatment of UV-B and oryzalin, low concentrations of ABA can reduce UV-B-induced root tip swelling, similar to taxol.

ABA Reduces UV-B-Induced Root Tip Expansion
To better understand the root morphology response to ABA and UV-B radiation, we compared the diameters of the root transition zone of seedlings exposed to taxol, oryzalin, ABA, and UV-B. Statistical analysis revealed that the root elongation inhibition (Figure 2b) and apical expansion caused by UV-B was significantly weakened by ABA application, while there was no significant difference between ABA concentrations of 0.25 μmol L −1 and 0.5 μmol L −1 (Figure 6). Compared with the treatment of UV-B and oryzalin, low concentrations of ABA can reduce UV-B-induced root tip swelling, similar to taxol. Figure 6. Effects of ABA and UV-B radiation on the root diameter of wild-type seedlings compared to those of taxol and oryzalin treatments. The diameters of the root transition zone of different treated seedlings were measured 24 h post-radiation by image processing software Image J. The data were statistically analyzed by data analysis software IBM SPSS Statistics 27. Untreated seedlings Figure 6. Effects of ABA and UV-B radiation on the root diameter of wild-type seedlings compared to those of taxol and oryzalin treatments. The diameters of the root transition zone of different treated seedlings were measured 24 h post-radiation by image processing software Image J. The data were statistically analyzed by data analysis software IBM SPSS Statistics 27. Untreated seedlings were used as control (CK). Each experiment was repeated three times. All the statistical analyses were conducted using the IBM SPSS Statistics 27 software. All values are expressed as the mean ± SE (n ≥ 27). And letters of a-d represent statistically significant groupings of p ≤ 0.05 based on a one-way ANOVA with the LSD multiple range tests. The results above indicated that both ABA and cortical microtubules are involved in the response to UV-B stress in roots. ABA stabilizes the transverse cortical microtubules and prevents UV-B-induced microtubule reorganization and consequent root tip swelling, which is beneficial for plants in enhancing resistance to UV-B stress.

Discussion
A. thaliana's roots have become an ideal model for research on plant architecture and developmental plasticity due to its simple structure and sensitivity to environmental stimuli [15,28]. As an underground organ of plants, roots have been ignored or even questioned about the scientific nature of their participation in UV-B response to aboveground light stress. Although generally unexposed to direct sunlight, plant roots also retain systematic signaling mechanisms in response to UV-B. Root UV-B sensitive 1 and 2 (RUS1 and RUS2) and four other RUS genes have been identified as contributing to the root UV-B signaling [9,31]. Seedling development in rus mutants is impeded under UV-B [9]. UV-B is perceived by the UVR8 protein in plants. The expression of UVR8 is also found in roots along with stems and leaves [10]. MYB transcription factors MYB73/77 regulate lateral root growth by interacting with UVR8 in roots in the presence of UV-B by hampering auxin signaling and hence combining UV-B and auxin pathways [12]. In addition, accumulating evidence indicates that root growth is influenced by light through the light-induced release of signaling molecules which can travel from the shoot to the root under natural conditions [28,[32][33][34]. Light was efficiently conducted through the stems to the roots in which ELONGATED HYPOCOTYL 5 (HY5) plays an important role. Photo-activated phytochrome B (phyB) can trigger the expression of HY5, a transcription factor, which promotes root growth in response to the expression of HY5 to promote photomorphogenesis [33,34]. Meanwhile, many other pathways are also involved in mediating light-regulated root and shoot growth and development, such as COP1-mediated light signaling, MYB73/MYB77-mediated UV-B signaling, and so on [35]. Thus studies on roots' response to UV-B stress at the morphological and physiological, cellular, and molecular levels have attracted increasing attention nowadays. Thus that is necessary to question the detailed cytological processes and mechanisms beneath root response to UV-B stress.
As a universal stress hormone, cellular ABA has been shown to mediate plants' development [15][16][17]30,[36][37][38]. ABA is synthesized and accumulated in roots and regulates primary root growth in A. thaliana in a concentration-dependent manner, which exogenously applied in low concentrations stimulates, while at high concentrations inhibits root growth [16,17]. ABA plays a vital role in equipping plants for environmental stresses. So, there is a probability of UV-B and ABA signaling crosstalk to manage the combinatorial effect of multiple environmental stresses, although the exact molecular mechanism is yet understudied. ABA biosynthesis mutants showed raised sensitivity toward UV-B stress, which reveals that ABA might be required for UV-B stress resistance. UV-B induces ABA accumulation, which increases the cytosolic Ca 2+ levels and activates nitric oxide signaling through nitric oxide synthase. The elevated nitric oxide signaling produces an enhanced antioxidant defense response [39]. An ABA-sensitive mutant sad2 (sensitive to ABA and drought) shows increased UV-B tolerance due to the absence of MYB4 protein in the nucleus and results in the accumulation of UV-absorbing pigments that shield the plant from UV-B radiation [20]. The present study analyzed the effects of endogenous and exogenous ABA in A. thaliana's response to UV-B stress. Results indicate that both endogenous accumulation and exogenous application of ABA can strengthen plants' adaptive response to UV-B stress (Figures 1, 2 and 6), which agrees with previous reports [5,20]. Interestingly, the possible role of the SAD2 gene functions not only as an importin mediating MYB4 nuclear trafficking [20] but also in the accumulation of ABA in response to UV-B light, which is supported by the fact that sad2-2 mutants exhibit excessive ABA accumulation on UV-B exposure (Figure 1c). However, how ABA strengthens plant tolerance to UV-B has yet to be characterized, and research into the role of the ABA signaling pathway in plant responses to UV-B is needed.
The microtubule cytoskeleton is widely involved in many key processes in plant morphogenesis [28,40] and the response to adverse environments [21][22][23]30] for the dynamic features, hence microtubule cytoskeletons are believed to perceive signal transduction in plant stress response [30,[41][42][43]. However, the crosstalk and underlying mechanism between microtubules and these signals are still insufficiently studied. In this study, we saw that UV-B radiation caused root tip ectopic outgrowths ( Figure 2) and alterations of cortical microtubules (Figure 3). And the microtubule alterations induced by UV-B tended to be attenuated by endogenous ABA. On the contrary, the alterations are sharpened in the ABA-deficient aba3 mutants with severe root tip expansion (Figure 4). Furthermore, similar to taxol treatment, exogenous application of low-concentration ABA significantly promoted the transverse cortical arrays ( Figure 5) to regulate root growth.
Cortical microtubule arrays are essential for determining the growth axis of the elongating cells in plants. Well-ordered transverse microtubule arrays promote cell elongation and restrict cell expansion, with some A. thaliana mutants harboring growth defects with unusual cortical arrays accordingly [44,45]. Therefore our results were consistent with previous reports that plants could harden, and ABA treatment increased the stability of tubulin microtubules and reduced the depolymerized action of oryzalin in winter wheat cultivars [46]. ABA signaling also partially rescues the oryzalin-induced (a microtubule-specific destabilizer) alternative root hydrotropism of wild-type A. thaliana [30].
While there are also some different results reported. Da Silva et al. [47] found that ABA treatment lowered the abundance of microtubules, altered their orientation, and inhibited embryo cell growth during coffee seed germination. Takatani et al. [42] found that 1 µmol L −1 ABA treatment induces ectopic outgrowths in epidermal cells of hypocotyls via promoting cortical microtubule disorganization. Furthermore, Jacques et al. [48] reported that UV-B radiation does not alter the cellular microtubule organization in A. thaliana leaves. However, the dynamic rearrangement of microtubules in response to UV-B is spontaneous and fast (Figure 3), which may not be observable over a few days. Besides that, it makes sense that our results differ from those reports due to different processes in different cells instead of root responses to UV-B.
Taken together, we propose that UV-B stress signals trigger endogenous ABA accumulation and cortical microtubule depolymerization, which alter the direction of root growth from elongation to isotropic expansion, as shown in Figure 7. Moderate increased ABA is necessary for protecting transverse cortical arrays to control root growth, which is an adaptive response for A. thaliana under ultraviolet-B stress. In addition, ABA accumulation and microtubule reorganization are two antagonistic mechanisms on root morphology, which are simultaneously activated and have significant effects on the plant response to UV-B stress. It's worth noting that both UV-B-induced root subapical expansion ( Figure 2) and alterations of cortical microtubules (Figure 3) are located in the transition zone, which is important to determine whether root cells will remain in the division zone or transit to elongation and differentiation. And this transition is dependent on precise gradients of phytohormones and the reorganization of microtubules [28]. By questioning the potential molecular mechanisms of the link between ABA and microtubules in A. thaliana roots re- It's worth noting that both UV-B-induced root subapical expansion ( Figure 2) and alterations of cortical microtubules (Figure 3) are located in the transition zone, which is important to determine whether root cells will remain in the division zone or transit to elongation and differentiation. And this transition is dependent on precise gradients of phytohormones and the reorganization of microtubules [28]. By questioning the potential molecular mechanisms of the link between ABA and microtubules in A. thaliana roots re- It's worth noting that both UV-B-induced root subapical expansion ( Figure 2) and alterations of cortical microtubules (Figure 3) are located in the transition zone, which is important to determine whether root cells will remain in the division zone or transit to elongation and differentiation. And this transition is dependent on precise gradients of phytohormones and the reorganization of microtubules [28]. By questioning the potential molecular mechanisms of the link between ABA and microtubules in A. thaliana roots re-. It's worth noting that both UV-B-induced root subapical expansion ( Figure 2) and alterations of cortical microtubules (Figure 3) are located in the transition zone, which is important to determine whether root cells will remain in the division zone or transit to elongation and differentiation. And this transition is dependent on precise gradients of phytohormones and the reorganization of microtubules [28]. By questioning the potential molecular mechanisms of the link between ABA and microtubules in A. thaliana roots response to UV-B, we further found that MAP18 (At5g44610) expression was regulated by ABA, which is supported by the TAIR database AtGenExpress Visualization Tool (AVT) ( Figure A2). The results showed that UV-B stress induced an irreversible inactivation of the quiescent center, which leads to the inhibition of cell proliferation and consequent growth of primary roots. At the same time, MAP18 plays an important role in UV-B-induced lateral root re-generation by regulating quiescent center activity (Figures A3-A6). All these facts will help us dissect and combine the roles of ABA, UV-B, and microtubule organization in roots in the future.
Author Contributions: F.S. and T.S. contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by L.S., and K.L., F.S. and T.S. analyzed the data and wrote the manuscript, and T.S. reviewed and edited the final manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.

Appendix A
Genes 2023, 14, x FOR PEER REVIEW 12 of 18 Appendix A Figure A1. Phenotype screening of Col-0 A. thaliana seedlings responding to different duration of UV-B radiation to determine that 30 min of 90 mJ cm −2 of UV-B radiation was selected for this study. Scale bars represent 1 cm. Figure A1. Phenotype screening of Col-0 A. thaliana seedlings responding to different duration of UV-B radiation to determine that 30 min of 90 mJ cm −2 of UV-B radiation was selected for this study. Scale bars represent 1 cm. Figure A1. Phenotype screening of Col-0 A. thaliana seedlings responding to different duration of UV-B radiation to determine that 30 min of 90 mJ cm −2 of UV-B radiation was selected for this study. Scale bars represent 1 cm. Figure A2. The AtGenExpress Visualization Tool (AVT) analysis of the TAIR database showed that the expression of microtubule-associated protein 18, MAP18, a microtubule destabilizing protein in A. thaliana (At5g44610), was regulated by ABA. Figure A2. The AtGenExpress Visualization Tool (AVT) analysis of the TAIR database showed that the expression of microtubule-associated protein 18, MAP18, a microtubule destabilizing protein in A. thaliana (At5g44610), was regulated by ABA.