Effect of water stress on cell division and cell-division-cycle 2-like cell-cycle kinase activity in wheat leaves

Abstract

In wheat (Triticum aestivum) seedlings subjected to a mild water stress (water potential of -0.3 MPa), the leaf-elongation rate was reduced by one-half and the mitotic activity of mesophyll cells was reduced to 42% of well-watered controls within 1 d. There was also a reduction in the length of the zone of mesophyll cell division to within 4 mm from the base compared with 8 mm in control leaves. However, the period of division continued longer in the stressed than in the control leaves, and the final cell number in the stressed leaves reached 85% of controls. Cyclin-dependent protein kinase enzymes that are required in vivo for DNA replication and mitosis were recovered from the meristematic zone of leaves by affinity for p13(suc1). Water stress caused a reduction in H1 histone kinase activity to one-half of the control level, although amounts of the enzyme were unaffected. Reduced activity was correlated with an increased proportion of the 34-kD Cdc2-like kinase (an enzyme sharing with the Cdc2 protein of other eukaryotes the same size, antigenic sites, affinity for p13(suc1), and H1 histone kinase catalytic activity) deactivated by tyrosine phosphorylation. Deactivation to 50% occurred within 3 h of stress imposition in cells at the base of the meristematic zone and was therefore too fast to be explained by a reduction in the rate at which cells reached mitosis because of slowing of growth; rather, stress must have acted more immediately on the enzyme. The operation of controls slowing the exit from the G1 and G2 phases is discussed. We suggest that a water-stress signal acts on Cdc2 kinase by increasing phosphorylation of tyrosine, causing a shift to the inhibited form and slowing cell production.

Figure 1

Increase in blade length of the first leaf of wheat in fully hydrated (unstressed; ○) and partially hydrated vermiculite at a water potential of −0.3 MPa (stressed; •). Treatment commenced at 24 h after imbibition. Bars show the ses of four experiments (n = 30).

Figure 2

Mitotic index of mesophyll cells as measured in longitudinal sections taken from the blade of the first leaf of wheat seedlings from unstressed (○) and stressed treatments (•) as defined in Figure 1 after 1 d (A), 2 d (B), or 4 d (C) of treatment. Bars show the ses (n = 32). The dotted line on B and C is the “developmental control,” i.e. the mitotic index of unstressed leaves of the same size as the stressed leaves (see text for further explanation).

Figure 3

Recovery of Cdc2-like kinase using p13^suc1 affinity chromatography with 20-μL p13 beads from extracts of leaf segments from unstressed (○, •) and stressed (□, ▪) plants after 48 h of treatment. A, Tissue 0 to 3 mm from leaf base. B, Tissue 3 to 6 mm from leaf base. Activity is related to milligrams of total extracted protein applied to p13 beads for purification prior to assay.

Figure 4

Activity of Cdc2-like kinase purified from extracts of leaf segments taken sequentially from the bases of the leaves of stressed and unstressed plants after 48 h of treatment.

Figure 5

Comparison of activity of Cdc2-like kinase purified from extracts of leaf segments taken from the basal 0 to 3 mm of leaves from unstressed and stressed leaves after 48 h of treatment with that purified from mixed extracts of the same tissues mixed in a 1:1 ratio. The broken line is the calculated mean of stressed and unstressed extracts. Bars show the ses (mean of three separate kinase assays).

Figure 6

Change with time in Cdc2-like kinase activity in leaves of plants that were first grown on fully hydrated vermiculite for 36 h and then transplanted to pots containing either fully or partially hydrated vermiculite at a water potential of −0.3 MPa (the transplantation protocol). Extracts were made from tissue 0 to 3 mm and 3 to 6 mm from the leaf base from unstressed and stressed plants and were purified before assay. •, Unstressed, 0 to 3 mm; ○, unstressed, 3 to 6 mm; ▪, stressed, 0 to 3 mm; and □, stressed, 3 to 6 mm. Bars show the average ses for all sampling times (mean of four assays).

Figure 7

Level of Cdc2-like protein in extracts from basal segments of stressed and unstressed leaf tissue after 48 h of treatment. A, Samples of total soluble protein separated by SDS-PAGE and transferred for western blotting, probed with PSTAIR antibody, and visualized with ¹²⁵I-labeled anti-rabbit IgG F(ab′)_2. B, Quantity of PSTAIR-containing 34-kD protein, in relative units measured by phosphor image analysis.

Figure 8

Frequency of mesophyll cell nuclei with DNA contents estimated by Feulgen stain, measured in longitudinal sections of leaves from unstressed and stressed plants after 48 h of treatment. A to C, Unstressed leaves. D to E, Stressed leaves. Mesophyll cells were scored in the basal 0- to 3-mm region of leaves (A and D) and the 3- to 6-mm region (B and E) and the 6- to 9-mm region of unstressed leaves only (C); cells in the 6- to 9-mm region of stressed leaves were entirely nonmitotic and arrested in G1 phase (not shown). Cells with DNA of 20 to 40 relative units were in G1 phase, and those with 45 to 70 units were in G2 phase. Open bars, Total nuclei; solid bars, nuclei in metaphase or anaphase.

Figure 9

Phospho-Tyr in 34-kD protein that was purified from 300-μg aliquots of total soluble protein by binding and elution with p13 beads. Alternate lanes carry purified enzyme from unstressed (U) and stressed (S) leaves taken from segments 0 to 3 mm (segt. 0–3) or 3 to 6 mm (segt. 3–6) from the base of the leaf. Plants were first grown on fully hydrated vermiculite for 36 h and then transplanted to pots containing either fully or partially hydrated vermiculite at a water potential of −0.3 MPa (the transplantation protocol). Plants were then sampled at 48 h (lanes 1–4) or 3 h (lanes 5–9). Lanes 1 through 8 were probed with anti-phospho-Tyr antibody and in lane 9 this antibody was precompeted (Comp) with 1 mm phospho-Tyr for 1 h before application to a duplicate of lane 8. A, Phosphor image of bound ¹²⁵I second antibody. B, Quantification of phospho-Tyr in 34-kD protein measured by phosphor image analysis.