Cyclin B1;1 activity is observed in lateral roots but not in the primary root during lethal salinity and salt stress recovery

ABSTRACT

Earlier, we reported that Arabidopsis young lateral roots (LR) exhibited improved lethal salinity tolerance as compared with the primary root (PR). We have shown that cell death processes which take place in the PR during salt stress are postponed in the LR. Still, very little is known about the regulation of cell survival mechanisms in the LR during salinity stress. Here we used transgenic Arabidopsis plants expressing Cyclin B1;1:GUS, to further study the responses to salinity in the PR and LR positions. We found strong Cyclin B1;1:GUS activity in young budding LR of salt stressed and stress recovered plants. The Cyclin B1;1:GUS activity dropped significantly in long LR and was almost completely abolished in the PR. Our data provides another line of evidence that position-dependent response occurs in Arabidopsis roots during lethal salinity. The possible roles Cyclin B1;1 plays in the young LR during the response to lethal salinity are discussed.

Results and discussion

Recently, we have shown in Arabidopsis that emerging and young lateral roots (LR) survive lethal salinity (200 mM NaCl) significantly longer than the primary root (PR).¹ We showed that autophagy mediated programmed cell death (A-PCD) and not apoptosis-like PCD (AL-PCD) takes place in the PR but not in the young LR. In addition, we found that as the young LR elongates, it loses its salt tolerance and tends to behave like the PR. Moreover, we have also shown that NADPH Oxidase activated ROS is required for the young LR improved salt tolerance.¹

Since the young LR remained alive during extended exposure to lethal salinity, we wanted to find whether acclimation and growth occurred in that position. Thus, we monitored root growth parameters in Arabidopsis WT (Col-0) young seedlings that were treated with 200 mM NaCl for several days. We found that no elongation occurred in the LR positions (Figure 1a and b). In addition, no new emergences of young LR primordia were detected (Figure 1c). Since non-lethal NaCl concentrations (75 and 150 mM) were reported to increase the number of new LR primordia,² the latter result implies that lethal salinity (200 mM NaCl) is mediated in the root differently.

Figure 1.

Growth inhibition of Arabidopsis roots during lethal salinity. Arabidopsis seedlings were grown on standard ½ MS medium for seven days, then the primary root (PR) and lateral root (LR) lengths was measured as well as the number of LR initiations. The seedlings were then transferred for additional 48h to standard ½ medium (control) or to standard medium supplemented with 200mM NaCl, followed by the same root parameters measurements. The following histograms present the change increase (in percent) between days 9 and 7, of the measured root parameters in the two treatments. (a) Percent increase in PR length, (b) Percent increase in LR length. (c) Percentage increase in new LR emergences. n=17 for (a) and (b) and n=27 for (c) S.E.

We still wondered whether some cell-cycle activities are maintained in the young LR during these salinity conditions. We thus used the reporter line Cyclin B1;1:GUS, which is an important cell cycle effector, known to positively regulate the G2-M transition.³ Previously, GUS activity of Cyclin B1;1 was shown to be eliminated from Arabidopsis LR meristems during non-lethal salt stress conditions (100 mM NaCl), indicating correlation between cell-cycle suppression and the temporal growth arrest which was observed in that position.⁴ Surprisingly, when we induced lethal salinity for 48 hours, strong Cyclin B1;1:GUS activity was detected in the young LR. The signal intensity in that position was slightly but insignificantly reduced as compared with the control signal (Figure 2, top row). The other root positions responded differently to the salt treatment: as compared with the control, a sharp reduction of the GUS signal was observed in the long LR (Figure 2, middle row), whereas elimination of the GUS signal occurred in the PR (Figure 2, lower row). The Cyclin B1;1:GUS signal was reexamined after salt stress when the seedlings were rescued and transferred to standard medium for additional 48 hours. During that time the Cyclin B1;1:GUS signal was maintained strongly in the young LR, slightly in the long LR, but remained absent from the PR (Figure 2 column c). These results provide another line of evidence supporting the vital staining assays we reported previously, which indicated that the LR but not the PR is still alive during the prolonged exposure to the lethal salt concentrations.

Figure 2.

Cyclin B1: GUS activity in the PR, long LR and short LR during lethal salinity and stress recovery. Seven days old Cyclin B1;1:GUS seedlings were grown on ½ MS (column A) or subjected to either 200 mM NaCl for 48 h (column B) or, removed after the salt period to ½ MS for two days (column C), followed by fixation and GUS staining. The top row, middle row and lower row represent the short LR, the long LR and the PR, respectively. The graph on the right side of each row represents the GUS quantification in those root types as integrated optical density (I.O.D). Images were captured by Inverted light microscope (Motic AE2000) as mentioned in methods. The seedlings were grown and treated as reported earlier.¹ GUS histochemical staining and quantification was done as described in the methods. The scale bars = 100 µm, n = 4 per each treatment, asterisks indicate statistical difference at p < .05 after performing Turkey Multiple comparison test. The experiment was replicated 3 times, with similar results.

It has been shown that in moderate salinity conditions, when LR growth cessation and cell cycle arrest occurs, Cyclin B1;1:GUS signal is eliminated.⁴ Nevertheless, here we show that when enduring growth arrest of the LR was induced by 200 mM NaCl (Figure 1), the Cyclin B1;1:GUS signal of young LR remained high. Since the strong Cyclin B1;1 activity we observed during the LR growth arrest probably does not reflect an active G2-M transition, we assume its presence may be connected to other roles Cyclin B1;1 can play. Recently, it was reported that Cyclin B1;1 also promotes DNA damage repair.^5,6 Thus, the strong LR Cyclin B1;1 activity we observed during extreme salt stress conditions might be involved in repair of DNA that is known to be damaged in Arabidopsis during salt stress.⁷

The exact role Cyclin B1;1 plays in the LR improved salt tolerance is still obscure. Future DNA repair experiments, combined with transcriptomic as well as proteomic approaches will likely shed more light on the ways Cyclin B1;1 is operated and orchestrated in the LR during lethal salinity tolerance. Such studies can potentially open novel gateways for improving salt stress tolerance in crop species which are predicted to face increased salinity in future.^8,9

Materials and methods

Plant material and growth

Arabidopsis thaliana (ecotype Columbia-0) Wild type and Cyclin B1;1:GUS reporter plants¹⁰ were used in this study. Seeds were plated on ½ MS Basal Salt medium (Caisson lab, USA) supplemented with 0.8% agar (Caisson lab, USA) under white light (120–130 µmol/m² sec) in a growth chamber with day/night cycles of 16 h at 24°C and 8 h at 18°C, with humidity of 40–60%. Plant were subjected to Salt stress 8 days after germination (DAG) by transferring then in sterile 30 mm petri dishes containing salt stress solution. Salt stress solution contained 200 mM NaCl was dissolved in ½ MS and pH was adjusted to 5.7 using 0.1 M KOH.

GUS staining, microscopy and image analysis

GUS staining of control and stressed Cyclin B1;1:GUS seedlings was performed as described earlier^11,12. In brief: seedling were washed and fixed in 90% acetone for 15 min under vacuum, rinsed twice with 10 mM phosphate buffer [pH 7] and incubated in GUS staining buffer (2 mM potassium ferricyanide, 2 mM potassium ferricyanide, 5 mM EDTA, 50 mM NaPO4, pH 7, 4 µl/ml of 0.25 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid, under vacuum for 15 min and kept for 2 hour at 37°C. Stained seedling were transferred to ethanol-acetic acid and cleared in Hoyer’s medium. Images were captured using inverted microscope AE2000 (Motic, Hong Kong) and Stereo Microscope SZ61 (Olympus, Japan) equipped with a Moticam 3+ camera. GUS staining quantification was done using Image J software following the method described earlier.^13,14 In brief, the image was transformed to Hue Saturation Brightness (HSB) mode, and the GUS stained area was measured in the Saturation channel. GUS The optical density of each root type was integrated with the area covered by GUS stained and present as integrated optical density (I.O.D)”

Funding Statement

This work was supported by Israel Science Foundation [ISF- UGC research grant no. 2713/16].

Disclosure of potential conflict of interest

No potential conflicts of interest were disclosed.