Inhibition of proteasome activity strongly affects kiwifruit pollen germination. Involvement of the ubiquitin/proteasome pathway as a major regulator

Abstract

The 26S proteasome is a multicatalytic complex that acts as primary protease of the ubiquitin-mediated proteolytic pathway in eukaryotes. We provide here the first evidence that the proteasome plays a key role in regulating pollen tube growth. Immunoblotting experiments revealed the presence of high levels of free ubiquitin and ubiquitin conjugates in rehydrated and germinating pollen of kiwifruit [Actinidia deliciosa var. deliciosa (A. Chev) C. F. Liang et A. R. Ferguson]. Proteasome activity, assayed fluorometrically, accompanied the progression of germination. Specific inhibitors of proteasome function such as benzyloxycarbonyl-leucinyl-leucinyl-leucinal (MG-132), clasto-lactacystin beta-lactone, and epoxomicin significantly decreased tube growth or altered tube morphology. High-molecular mass, ubiquitinated proteins accumulated in MG-132- and beta-lactone-treated pollen, indicating that proteasome function was effectively impaired. The inhibitors were also able to decrease in vitro proteasome activity in pollen extracts. Because MG-132 can inhibit calpains, as well as the proteasome, trans-epoxy succinyl-L-leucylamido-(4-guanidino) butane (E-64), an inhibitor of cysteine proteases, was investigated. Some reduction in tube growth rate was observed, but only at 80 microM E-64, and no abnormal tubes were produced. Furthermore, no inhibition of tube growth was observed when another inhibitor of cysteine proteases, leupeptin, or inhibitors of serine and aspartic proteases (phenylmethylsulfonyl fluoride and pepstatin) were used. Our results indicate that protein turnover during tube organization and elongation in kiwifruit pollen is important, and our results also implicate the ubiquitin/26S proteasome as the major proteolytic pathway involved.

Figure 1

Time course of kiwifruit pollen tube growth in standard medium (●) and in the presence of 300 μm cycloheximide (▵). Growth is expressed as A₅₀₀ of sonicated aqueous suspension of grains/tubes measured at 1-h intervals. Slope of control regression is 0.107 ± 0.005. Data are the means ± sd of three independent experiments.

Figure 2

Distribution of free ubiquitin and ubiquitin conjugates in quiescent (Q), rehydrated (R), and germinating (15–180 min of incubation) kiwifruit pollen. A, B, and D, Twenty micrograms of protein was electrophoresed on a 10% (w/v) polyacrylamide gel and stained with Coomassie Blue (A) or electroblotted onto nitrocellulose membrane and probed with an affinity-purified polyclonal anti-ubiquitin antibody (B) or an anti-actin antibody (D). C, Immunoblot detection of free ubiquitin. Five micrograms of protein was electrophoresed on a 14% (w/v) polyacrylamide gel. The nitrocellulose membrane was probed as reported above. The positions of molecular mass markers (in kilodaltons) are shown on the left.

Figure 3

Proteasome activity in quiescent and germinating kiwifruit pollen. Extracts of ungerminated or germinated pollen were incubated in the presence of 200 μm sLLVY-NH-Mec at 30°C. The breakdown of the fluorogenic peptide was monitored by a fluorescence spectrophotometer for 5 min. A, Proteolytic activity in the presence (white columns) or absence (gray columns) of 2 mm ATP. B, Proteolytic activity in 30-min-germinated pollen extract as affected by the proteasome inhibitors MG-132 (50 μm), clasto-lactacystin β-lactone (10 μm), or epoxomicin (5 μm). Data are means ± sd of three measurements.

Figure 4

Effect of proteasome inhibitors on kiwifruit pollen tube growth over time. Growth is expressed as A₅₀₀ of a sonicated aqueous suspension of grains/tubes measured at 1-h intervals. A, Effect of MG-132 on tube growth. Slope of DMSO control (●) regression is 0.129 ± 0.007; slope of 40 μm MG-132-treated (▵) regression is 0.058 ± 0.005; and slope of 80 μm MG-132-treated (□) regression is 0.022 ± 0.006. B, Effect of 10 μm clasto-lactacystin β-lactone on tube growth during the first 2 h and during the following 3 to 4.5 h of incubation (inset). Slope of DMSO control (●) regression over total 4.5 h of incubation is 0.099 ± 0.003 and slope of β-lactone-treated (▵) regression during 3 to 4.5 h of incubation is 0.016 ± 0.002. C, Effect of epoxomicin on tube growth. Slope of DMSO control (●) regression is 0.107 ± 0.001; slope of 1 μm epoxomicin-treated (▵) regression is 0.081 ± 0.008; and slope of 5 μm epoxomicin-treated (□) regression is 0.068 ± 0.004. Data are the means ± sd of three independent experiments.

Figure 5

Abnormal morphology of kiwifruit pollen tubes induced by MG-132 treatment. A, Three hours of incubation in the presence of 40 μm MG-132; bar = 30 μm. B, Threee hours of incubation in the presence of 80 μm MG-132; bar = 30 μm. C, Untreated pollen tubes after 3 h of incubation in standard medium; bar = 100 μm. D, Untreated pollen tubes (controls) after 3 h of incubation in standard medium containing DMSO solvent; bar = 100 μm.

Figure 6

Reversion of the inhibitory effect of MG-132 on kiwifruit pollen tube growth. A, Pollen tube growth over time. DMSO controls (□) and 80 μm MG-132-treated pollen (○) were collected and washed after 1 h of incubation (arrow) and were transferred to fresh standard medium containing DMSO. Another lot of 80 μm MG-132-treated pollen (●) was incubated in the presence of the inhibitor throughout. Growth was monitored by measuring tube length (micrometers) as described in “Materials and Methods.” Data are the means ± sd (n = 300). B, Pollen treated for 1 h with 80 μm MG-132, 45 min after transfer to inhibitor-free medium. C, DMSO controls, treated as described above, 45 min after transfer to fresh medium containing DMSO. D, Pollen after 105 min of incubation in the presence of 80 μm MG-132.

Figure 7

Effect of non-proteasomal protease inhibitors on kiwifruit pollen tube growth over time. Growth is expressed as A₅₀₀ of a sonicated aqueous suspension of grains/tubes measured at 1-h intervals. A, Effect of E-64 on tube growth. Slope of control (●) regression is 0.102 ± 0.002; slope of 40 μm E-64-treated (▵) regression is 0.108 ± 0.009; and slope of 80 μm E-64-treated (□) regression is 0.087 ± 0.005. B, Effect of leupeptin on tube growth. Slope of control (●) regression is 0.099 ± 0.006 and slope of 50 μm leupeptin-treated (▵) regression is 0.101 ± 0.005. C, Effect of pepstatin on tube growth. Slope of ethanol control (●) regression is 0.089 ± 0.006 and slope of 50 μm pepstatin-treated (▵) regression is 0.093 ± 0.005. D, Effect of PMSF on tube growth. Slope of ethanol control (●) regression is 0.084 ± 0.007; slope of 50 μm PMSF-treated (▵) regression is 0.086 ± 0.006; and slope of 100 μm PMSF-treated (□) regression is 0.085 ± 0.009. Data are the means ± sd of three independent experiments.

Figure 8

Effect of proteasome inhibitors on accumulation of high-molecular mass ubiquitin-conjugated proteins in germinating kiwifruit pollen. A and C, Immunoblotting of total protein (20 μg per lane) extracted from pollen incubated with 40 μm MG-132, 80 μm E-64, or 10 μm β-lactone for different times and from pollen incubated in the medium without the respective inhibitor. Total protein was electrophoresed on 10% (w/v) polyacrylamide gels and was immunoblotted using polyclonal anti-ubiquitin antibody (A) or an anti-actin antibody (C). B, Immunoblot detection of free ubiquitin (each lane was loaded with 5 μg of protein). Molecular mass of standard proteins are indicated on the left (in kilodaltons).