Six cellulase genes were isolated from total RNA of the ethylenetreated tomato (Lycopersicon esculentum Mill.) flower abscission zone by reverse-transcription polymerase chain reaction using degenerate primers to conserved amino acid sequences from known plant cellulases. Four of the gene fragments are homologous to fruit pericarp cellulases. l h e other two are nove1 cellulase genes, referred to as Ce15 and Ce16. Breakstrength and cellulase gene expression were then analyzed in naturally abscising flowers and flower explants. I n both naturally abscising flowers and flower explants induced to abscise in air or ethylene, both new cellulase mRNAs were correlated with flower shedding. Whereas the Ce15 mRNA increased in later stages of abscission, the Ce16 mRNA was present i n nonabscising flowers and then decreased i n the final stage of abscission. A third cellulase, Cell, increased during the final stage of abscission in flower explants and yet did not increase during shedding i n planta, although it was detectable at low levels in all abscission stages. Cell and Ce15 mRNA decreased 99% when indole-3-acetic acid was added during ethylene treatment, consistent with low levels of abscission (3%). I n contrast, Ce16 mRNA increased slightly when indole-3-acetic acid was added. These results suggest that abscission is a multistep process involving both activated and repressed cellulase genes and that the relative importance of each cellulase in the process depends on the physiological conditions under which abscission takes place.Abscission is a process that allows plants to shed an organ from the parent body in a controlled fashion. Separation results from catabolic alterations in the middle lamellae and the primary cell wall in a few layers of specialized cells usually located at the base of the organ to be shed (Sexton and Roberts, 1982). The breakdown of cell-wall components in these specialized cells is so complete that after separation the cells of the scar surface look rounded, as if they were protoplasts (Sexton et al., 1989), and yet the destruction is gradual and spatially confined. These specialized cells have a unique ability to sense and respond to abscission signal(s) by initiating a gene expression program that causes separation (Sexton and Roberts, 1982). The enzymatic machinery engaged by the abscission program operates through severa1 classes of enzymes including endo-P-(1,4)-glucanases, commonly referred to as cellulases (Lewis and Varner, 1970; de1 Campillo et al., 1990),

KORRIGAN1 (KOR1) is a membrane-bound cellulase implicated in cellulose biosynthesis. PttCel9A1 from hybrid aspen (Populus tremula L. x tremuloides Michx.) has high sequence similarity to KOR1 and we demonstrate here that it complements kor1-1 mutants, indicating that it is a KOR1 ortholog. We investigated the function of PttCel9A1/KOR1 in Arabidopsis secondary growth using transgenic lines expressing 35S::PttCel9A1 and the KOR1 mutant line irx2-2. The presence of elevated levels of PttCel9A1/KOR1 in secondary walls of 35S::PttCel9A1 lines was confirmed by in muro visualization of cellulase activity. Compared with the wild type, 35S::PttCel9A1 lines had higher trifluoroacetic acid (TFA)-hydrolyzable glucan contents, similar Updegraff cellulose contents and lower cellulose crystallinity indices, as determined by (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy. irx2-2 mutants had wild-type TFA-hydrolyzable glucan contents, but reduced Updegraff cellulose contents and higher than wild-type cellulose crystallinity indices. The data support the hypothesis that PttCel9A1/KOR1 activity is present in cell walls, where it facilitates cellulose biosynthesis in a way that increases the amount of non-crystalline cellulose.

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The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACI). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACI codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)' RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACI decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACI or expression of cellulase activity. point (pI 9.5, subsequently referred to as 9.5 cellulase) (6). This 9.5 cellulase was purified and used to prepare cellulase specific antiserum (6). Durbin et al. (6) used a sensitive radioimmune assay to show a close correlation between the appearance of the 9.5 cellulase protein and the onset of abscission. Sexton et al. (27) extended this correlation to show that 9.5 cellulase is predominantly associated with a two to three cell wide separation layer though low levels of activity were also found in adjacent stem and petiolar tissue.Leaf abscission can be induced in the petiole by removal of the subtending leaf blade. Jackson and Osborne (13) Treatment of Plant Materials. Bean (Phaseolus vulgaris cv Red Kidney) plants were grown in a greenhouse and harvested 12 d after germination at which time primary leaves are fully expanded. Explants were prepared by cutting the stem below the cotyledons and then removing the cotyledons. The leaf blades of the primary leaves were removed from explants by cutting 2 cm above the basal pulvinus. The unexpanded trifoliate leaves above the first node were also detached from the explants. Explants were then surface sterilized with a 2 min bath in 0.5% sodium hypochlorite (bleach), rinsed with several volumes of water, and placed upright in styrofoam cups filled with distilled water. Cups containing the explants were placed into a 22 L chamber and the explants exposed to 5 gL/L ethylene in air at a flow rate of 2 L/min. Where indicated, 5 x 1o-6 M IAA in lanolin was applied onto the distal petiolar stump of explants 4 h prior to ethylene treatment. In experim...

The sloughing of root cap cells from the root tip is important because it assists the growing root in penetrating the soil. Using a promoter-reporter (GUS) and RT-PCR analysis, we identified an endo-beta-1,4-glucanase (AtCel5) of Arabidopsis thaliana that is expressed exclusively in root cap cells of both primary and secondary roots. Expression is inhibited by high concentrations of IAA, both exogenous and internal, as well as by ABA. AtCel5 expression begins once the mature tissue pattern is established and continues for 3 weeks. GUS staining is observed in both root cap cells that are still attached and cells that have already been shed. Using AtCel5-GUS as a marker, we observed that the root cap cells begin to separate at the sides of the tip while the cells of the central region of the tip separate last. Separation involves sequential tiers of intact cells that separate from the periphery of the root tip. A homozygous T-DNA insertion mutant that does not express AtCel5 forms the root cap and sheds root cap cells but sloughing is less efficient compared to wild type. The reduction in sloughing in the mutant does not affect the overall growth performance of the plant in loose media. The modest effect of abolishing AtCel5 expression suggests that there are multiple redundant genes regulating the process of sloughing of the root cap, including AtCel3/At1g71380, the paralog of the AtCel5 gene that is also expressed in the root cap cells. Thus, these two endo-1,4-beta-D-glucanases may have a role in the sloughing of border cells from the root tip. We propose that AtCel5, provides a new molecular marker to further analyze the process of root cap cell separation and a root cap specific promoter for targeting to the environment genes with beneficial properties for plant growth.

No abstract

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of fl-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatinlike protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (pl4)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.known, however, that RNA and protein synthesis are required for abscission to occur (2); after ethylene treatment, abscission zones respond with the induction and accumulation of new proteins (12, 13); and after fracture, the cells of the scar surface, when viewed under scanning electron microscopy, appear rounded as if they were protoplasts (21). Thus, abscission involves the expression, synthesis, and secretion of hydrolytic enzymes in a highly coordinated manner. In bean, a cellulase, referred to as 9.5 cellulase, has been implicated in abscission. This cellulase is induced by ethylene and accumulates to high levels in the abscission zone (25). The increase in activity of 9.5 cellulase has been correlated...

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.