The experimental herbicide CGA 325'615 inhibits synthesis of crystalline cellulose and causes accumulation of non-crystalline beta-1,4-glucan associated with CesA protein

Abstract

Developing cotton (Gossypium hirsutum) fibers, cultured in vitro with their associated ovules, were used to compare the effects of two herbicides that inhibit cellulose synthesis: 2,6-dichlorobenzonitrile (DCB) and an experimental thiatriazine-based herbicide, CGA 325'615. CGA 325'615 in nanomolar concentrations or DCB in micromolar concentrations causes inhibition of synthesis of crystalline cellulose. Unlike DCB, CGA 325'615 also causes concomitant accumulation of non-crystalline beta-1,4-glucan that can be at least partially solubilized from fiber walls with ammonium oxalate. The unusual solubility of this accumulated glucan may be explained by its strong association with protein. Treatment of the glucan fraction with protease changes its size distribution and leads to precipitation of the glucan. Treatment of the glucan fraction with cellulase digests the glucan and also releases protein that has been characterized as GhCesA-1 and GhCesA-2--proteins that are believed to represent the catalytic subunit of cellulose synthase. The fact that cellulase treatment is required to release this protein indicates an extremely tight association of the glucan with the CesA proteins. In addition, CGA 325'615, but not DCB, also causes accumulation of CesA protein and a membrane-associated cellulase in the membrane fraction of fibers. In addition to the effects of CGA 325'615 on levels of both of these proteins, the level of both also shows coordinate regulation during fiber development, further suggesting they are both important for cellulose synthesis. The accumulation of non-crystalline glucan caused by CGA 325'615 mimics the phenotype of the cellulose-deficient rsw1 mutant of Arabidopsis that also accumulates an apparently similar glucan (T. Arioli, L. Peng, A.S. Betzner, J. Burn, W. Wittke, W. Herth, C. Camilleri, H. Hofte, J. Plazinski, R. Birch et al. [1998] Science 279: 717).

Figure 1

The effect of CGA 325′615 on incorporation of [U-¹⁴C]Glc into cell wall material in cultured cotton ovules with their associated fibers. The experiment was conducted using 21 DPA fibers and the incubation time in labeled Glc with or without herbicide was 4 h. Crystalline cellulose is defined as that material resistant to digestion by acetic-nitric reagent (Updegraff, 1969); radioactivity solubilized by this treatment is represented as the non-crystalline wall material. Each data point represents the average of triplicate samples.

Figure 2

Comparison of the effect of CGA 325′615 and DCB on cell wall synthesis. The experiment was conducted as described in “Materials and Methods” and in the legend to Figure 1. The concentration of CGA 325′615 was 10 nm and the concentration of DCB was 25 μm.

Figure 3

Non-crystalline wall material solubilized by sequential extraction of cotton fiber cell walls. CGA-treated fibers had been incubated for 4 h in 10 nm CGA 325′615. Details of cell wall preparation and extractions are given in “Materials and Methods.” DMSO, Dimethylsulfoxide; CM, chloroform:methanol; ACID, material solubilized by acetic-nitric treatment following KOH extraction.

Figure 4

Sugar composition and linkage analysis of AO fraction. CGA treatment prior to wall analyses was for 4 h at 10 nm CGA 325′615. A, Monosaccharide composition as determined by GLC of radioactive alditol acetate derivatives. B, Analysis of partially methylated, partially acetylated radioactive derivatives by GLC. “Others” refers to the sum of radioactivity in all other derivatives, none of which was significantly enhanced by CGA 325′615.

Figure 5

Glucanase treatments alter the size distribution of the radioactive material in the AO fraction. Chromatography was on Superdex 200 using 0.1% (w/v) AO as solvent. The fraction was obtained from walls of fibers treated for 4 h with 10 nm CGA 325′615 and was applied to the column untreated or following digestion with exo-1,3-β-glucanase or endo-1,4-β-glucanase as detailed in “Materials and Methods.” Equal amounts of radioactivity (cpm) were loaded for control and treated samples. Vo represents the void volume of the column, and Vt the position where totally included molecules such as Glc and cellobiose would elute.

Figure 6

Protease treatment alters the size distribution of the radioactive material in the AO fraction. All conditions were as in the legend to Figure 5, except that digestion was carried out by treatment with Proteinase K as detailed in “Materials and Methods.” Equal amounts of radioactivity (cpm) were loaded for control and treated samples.

Figure 7

Characteristics of glucan that precipitates from the AO fraction following digestion with protease and chilling. A, The precipitate stains in fluorescence microscopy with the glucan-binding dye Calcofluor. B, Binding on polyvinylidene difluoride membrane of the insoluble glucan from CGA 325′615-treated fibers to a cellulose-binding protein (CBD) detected by probing with anti-CBD antibody (top), and an autoradiogram showing that this glucan retains its radioactivity upon precipitation (bottom).

Figure 8

Methylation analysis of protease-treated glucan that precipitates upon chilling. A, GLC trace showing elution patterns for standards. a, t-Glc; b, 3-Glc; c, 4-Glc; and d, inositol. B, GLC trace of the partially methylated, partially acetylated derivatives from the precipitated glucan. The major peak (c) runs coincident with the 4-Glc standard. Peak a represents retention time for t-Glc, whereas peak a′ represents an unidentified terminal hexose, and peak d is the inositol standard. C, Mass spectrum of peak c that contains ions diagnostic of 4-Glc (indicated by asterisks).

Figure 9

Western blot of proteins derived from the AO fraction of fibers incubated without (control) or with 10 nm CGA 325′615 or 25 μm DCB. Protein was detected by use of an antibody directed against the zinc-finger domain of GhCesA-1. EC, Endocellulase enzyme alone. AO samples not treated (−) or treated (+) with endocellulase prior to electrophoresis. Protein levels were too low to quantify accurately, and so equivalent volumes of the AO fractions from the same number of control or herbicide-treated ovules/fibers were loaded. This represented <2 μg of protein in both cases.

Figure 10

Changes in level of CesA and Cel3/Kor proteins in CesA and cellulase (Kor) proteins during development or in response to herbicide treatment. A, Cultured ovules with fibers were incubated for 4 h without herbicide or with 10 nm CGA 325′615 or 25 μm DCB prior to isolation of crude microsomal membranes. Ten micrograms of membrane protein was loaded per lane. The top lane was probed with the antibody against the zinc-finger domain of GhCesA-1. The bottom lane was probed using antibody against the tomato Cel3 (Kor) protein. B, Western blot performed as above but showing changes in level of CesA and Cel3/Kor protein in crude microsomal membranes (10 μg/lane) derived from plant-grown fibers during cotton fiber development. Ten d post-anthesis represents stage of primary wall synthesis; 17 DPA transition to secondary wall synthesis, and fibers at 24 DPA are fully engaged in secondary wall synthesis.