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The occurrence of enzymes associated with bean leaf abscission was investigated in bean (Phaseolus vulgaris) flower reproductive organs in which catabolic cell wall events are essential during anther and pistil development. Cellulase activity was detected in high levels in both pistil and anthers of bean flowers before anthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified a protein in anthers and pistil with the same size (51 kilodaltons) and serologically closely related to the abscission cellulase. The accumulation of 9.5 cellulase protein in the anther is developmentally regulated and increases from undetectable levels at very young stages of anther development to high levels as the anther matures. In the pistil, the 9.5 cellulase was localized in the upper part of the pistil where the stigma and the stylar neck reside and was detected in the youngest developmental stage analyzed. Antibodies against basic chitinase, which accumulates to high levels in abscission zones after exposure to ethylene, identified a protein with the same size (33 kilodaltons) and serologically closely related, in both anthers and upper portion of the pistil. In contrast, a 45-kilodalton protein and the basic beta-1,3-glucanase associated with abscission were undetected in bean reproductive organs. Interestingly, beta-1,3-glucanase activity was detected in young bean anthers and decreased at anthesis, but the anther beta-1,3-glucanase is serologically unrelated to the basic beta-1,3-glucanase. Thus, it appears that the basic cellulase and chitinase occur in combination in many plant processes that require major cell wall disruption, whereas hemicellulases such as beta-1,3-glucanase are specific to each process.

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 β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like 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 (p14)-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.

A fundamental event in abscission is the breakdown of cell wall material in a discrete zone of cells known as the separation layer. Three dimensional images produced by viewing tissue prints of abscission zones on nitrocellulose (NC) membranes with incident illumination showed changes in the tissue integrity taking place in the separation layer as the process of abscission proceeded. The cell softening which occurs due to the dissolution of the cell wall appeared in the tissue prints as a diffuse line at the anatomical transition between the pulvinus and petiole and was easily observed on NC tissue prints of either longitudinal or serial cross-sections through abscission zones. In bean leaf abscission the dissolution of cell walls has been correlated with the appearance of a form of cellulase with an isoelectric point of pH 9.5. Antibodies specific for this enzyme were used to study the localization of 9.5 cellulase in the distal abscission zone of Phaseolus vulgaris L., cv Red Kidney after tissue printing on NC. It was found that 9.5 cellulase was localized in the separation layer but also occurred in the vascular tissue of the adjacent pulvinus. No antibody binding was observed in nonabscising tissue or preimmune controls. These results confirm previous biochemical studies and demonstrate that immunostaining of nitrocellulose tissue prints is a fast and reliable method to localize proteins or enzymes in plant tissue.

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 beta-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like 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 (p14)-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.

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.

Lymphocyte subset analysis was performed on 114 healthy children and 84 healthy adults. Samples were prepared by a whole blood lysis technique and analyzed by flow cytometry. The percentage and total number of CD3+, CD4+, CD8+, and CD19+ lymphocytes were calculated for each of six age groups. A direct correlation with age was seen in the percentages of CD3+, CD4+, and CD8+ lymphocytes. The absolute number and percentage of total lymphocytes, the percentage and absolute number of CD19+ lymphocytes, and the absolute number of CD3+ lymphocytes decreased with age. No significant correlation with age was observed for white blood cells, the absolute number of CD4+ and CD8+ lymphocytes, and the CD4+/CD8+ ratio.

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10(-5) or 10(-2)m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10(-2)m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10(-5)m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.

Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10- to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which cellulase was associated had a peak equilibrium density of 1.16 to 1.17 g/cm(3) which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate cellulase was associated with the plasma membrane.

1. BURiK, L. G. ANI) T. (C. lrso. 1958. E1 ffects ot gibberellic acid on N'icotiacwa plants. Nature 181: 1672-73. 2. FRANKLAND, B. ANI) P. F. WARING. 1960. Effect of gibberellic acid oni the hypocotyl growth of lettuce seedlings. Nature 185: 225. 3. HALEVY, A. H., S. P. MONSELISE, AND Z. PLAUT. 1964. Effect of gibberellin on translocation and on dry matter and water content in several plant species. Physiol. Plantarum 17: 49-62. 4. LALORAYA, M. M. AND V. K. RAI. 1962. Promotion of hypocotyl growth with gibberellic acid in epigeally growing seedlings. Physiol. Plantarum 15: 649-55. 5. NOODEN, L. D. AND K. V. THIMANN. 1963. Evidence for a requirment for protein synthesis for auxin-induced cell enlarbmiienit. Proc. Natl. Acad. Sci., U.S.A., 50: 194-200. 6. PALEG, L., D. ASPINALL, B. COOMBE, AND P. NICHOLLS. 1964. Plhysiological effects of gibberellic acid VI. Other gibberellins in three test systems. Plant Physiol. 39: 286-89. 7. RAI, V. K. AND M. M. LALORAYA. 19f63. Studies on changes in ribonucleic acid associate(d wvith gibberellin induced hypocotyl growtlh in lettuce seedlings. Proc. 50th Imld. Sci. Conig. Part III, Abs., p 413. 8. THIMANN, K. V. AND G. M. Loos. 1957. Proteini synthesis during water uptake by tuber tissue. Plant Physiol. 32: 274-79. 9. THIMANN, K. V. AND M. M. LALORAYA. 1960. Changes in nitrogen in pea stem sections under the action of kinetin. Physiol. Plantarum 13: 16578. 10. YABUTA, T., Y. SIUMNIKI, AND T. TAKAHASHI. 1943. Biochemistry of bakanae fungus of rice. Part 16. The effects of gibberellin on special components and special tissues of plants. (4) Action of gibberellin on tobacco seedlings. J. Agr. Chem. Soc. Japan. 19: 396. 11. YABUTA, T., Y. STAMIKI, K. FUKITNAGA, AND M. HORIUCHI. 1951. Biochemistry of hakanae fungus. Part 22. Chemical composition of rice seedlings treated with gibberellin (2). J. Agr. C,hem. Soc. Japan. 24: 396-97.

idea of measuring vital capacity, breathing capacity or chest circumference, then expan sion. This measure was usually added to the weight/height index. Vierordt's formula for determining weight in connection with body length and chest circumference endeavored to present this combination mathematically. Some investigators believed that strength was a factor in physical well-being, and intro duced the measures of grip, generally used with other measures. Naccarati evolved the

The ultrastructural reversion of chromoplasts to chloroplasts in regreening Valencia oranges is described. During reversion of chromoplasts to chloroplasts the grana-fretwork system is evidently built up from small vesicles which pinch off from the inner plastid membrane. As the chromoplast regreens, the large osmiophilic globules become reduced in size and number, and there may be a utilization of the lipid material of these globules during the formation of the internal membrane system. It is suggested that irreversible senescences of the chromoplasts probably is dependent on changes in the plastid membrane.

Debladed midribs of citrus leaves showed the typical delay of abscission in response to indoleacetic acid (IAA), and the typical acceleration of abscission in response to gibberellic acid (GA). Interaction experiments with these 2 hormones indicated that the balance of the 2 hormones may be more important in regulating abscission than the quantity of either. The often reported acceleration of abscission with low quantities of IAA did not seem to exist in citrus. IAA did accelerate abscission in this tissue when its application was delayed for at least 24 hours after deblading, which suggests the 2-stage effect is also present in citrus.When abscission was first delayed with IAA and then allowed to continue, the rate of abscission proceeded at a slower rate than was typical for this tissue. This slower rate was also typical of the effect observed when GA overcame the abscission retarding effect of IAA. The phenylurethane, Barban, blocked the GA acceleration of abscission, but it did not affect the rate of abscission of control or IAA treated midribs.

Evidence for the non-indolic nature of the new citrus auxin is presented on the basis of fluorometric properties, thin-layer chromatography, Ehrlich's colour reaction, paper electrophoresis, and the infra-red spectra determinations. Citrus auxin had a lower Rf in TLC than IAA, did not give the typical indole reaction with Ehrlich's reagent, and behaved differently in electrophoresis. The infra-red spectra also provided preliminary information concerning chemical structure. The hypothesis that indolic compounds constitute the only natural auxins in higher plants should be revised in view of this evidence that a non-indole auxin exists in higher plants.

In Southern California the Valencia orange (Citrus sinensis, L.) attains maximum orange rind color during winter and regreens somewhat by harvest time the following summer. The degree of regreening has been correlated with temperature patterns and with nitrogen nutrition (2, 5). It has also been reported (3) that applications of potassium gibberellate (KGA) to Valencia trees were associated with enhanced regreening of the fruit. The objective of this investigation was to obtain additional information about the influence of KGA on regreening of the Valencia orange fruit.

A gradient elution column chromatography technique and a step-wise technique succeeded in differentiating between IAA and the citrus auxin. IAA was eluted ahead of the citrus auxin in both systems. The highest Avena curvature ever obtained from the citrus auxin occurred after the auxin had passed through the 2 purification techniques and a paper chromatography step. This is probably due to the elimination of inhibitors. Fluorometric assay, Ehrlich's reaction, thin-layer chromatography, and biological assay were used for the detection of IAA or citrus auxin in the column eluates.

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 (pBACl). 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 pBACl 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 pBACl 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 pBACl or expression of cellulase activity.

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10(-5) or 10(-2)m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10(-2)m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10(-5)m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.

Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10- to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which cellulase was associated had a peak equilibrium density of 1.16 to 1.17 g/cm(3) which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate cellulase was associated with the plasma membrane.

Plasma membranes from abscission zones of kidney bean seedlings were isolated on sucrose density gradients using cellulase and KCl-stimulated ATPase as marker enzymes. Following treatment of the seedling explants with 50 microliters per liter ethylene, an increase in the buoyant density of the plasma membrane fraction from 1.165 to 1.175 grams per cubic centimeter was observed. Such a change is consistent with the loss of lipids from the senescent cells of the abscission zone.

Cellulase (beta-1, 4-glucan-glucanohydrolase EC 3.2.1.4) activity in the abscission zone of red kidney bean (Phaseolus vulgaris) was previously shown to exist in at least two different molecular forms. The form of the enzyme which has an isoelectric point of 4.5 is present in both abscising and nonabscising tissue and requires grinding for extraction. Another form of the enzyme which has an isoelectric point of 9.5 is present only in tissue in which the abscission process has been induced. Further, much of this form of cellulase can be removed from the tissue by vacuum infiltration with buffer. Time course studies indicate that while the increase in measurable cellulase activity in tissue which is actively undergoing abscission was due primarily to the appearance of cellulase 9.5, this form of the enzyme cannot be removed by vacuum infiltration until after the breakstrength of the abscission zone has decreased nearly to zero. The intracellular localization of these two forms of cellulase is discussed.

When abscission in leaf explants from Phaseolus vulgaris, cultivar Red Kidney, was allowed to proceed while the explants were in (2)H(2)O, a 1.25% increase in the buoyant density of cellulase in a cesium chloride gradient was observed. These data indicate that the increase in cellulase activity during abscission is a result of the synthesis of new protein. Two differentially soluble forms of cellulase are present in the abscission zone. The form which is soluble only in a high salt buffer seems more closely related to the abscission process than the form which is soluble in dilute buffer. The correlation between changes in pull force and increase in cellulase activity and the effects of several hormones on cellulase activity are discussed.

Red light, which produces the physiologically active form of phytochrome (Pfr), inhibited epicotyl elongation in intact dark-grown Alaska pea seedlings. This red light response was detectable 3 hours after the light treatment and became pronounced after 5 hours. The growth inhibition was completely reversed by far red light applied immediately after the red or by pretreatment of the seedlings with the plant hormone gibberellin A(3).Comparison of the total (32)P-labeled nucleic acids from control and red light-treated Alaska pea epicotyls on methylated albumin-kieselguhr columns revealed a marked alteration of the pattern of nucleic acid synthesis in this plant material with little or no effect on total isotope incorporation into nucleic acids. A single 5-minute red light perturbation caused a 2-fold stimulation of (32)P incorporation into the tRNA fraction while, simultaneously, (32)P incorporation into tenaciously bound RNA was reduced to 50% of control levels. Red light treatment had no effect on (32)P incorporation into the DNA-RNA, rRNA, or mRNA fractions. Far red light reversed the effect of red light on tRNA synthesis but did not restore tenaciously bound RNA levels to the control value. Gibberellin A(3) treatment did not cause reversal of any of the red light effects on RNA synthesis.These light-induced changes in nucleic acids were measurable before any changes in the physiological response (epicotyl elongation) could be detected. These results are consistent with a phytochrome-mediated differential gene activation mechanism in the Alaska pea epicotyl elongation system.

Abscission zone tissue of citrus was shown to have a higher rate of protein synthesis than tissue distal or proximal to it, based on the incorporation of leucine-1-(14)C. The proximal tissue had the slowest rate of protein synthesis. As the tissue approached abscission, the rate of synthesis in the abscission zone decreased and the differences in rate of protein synthesis between the 3 zones almost disappeared. IAA, which delayed abscission, maintained the protein synthesis gradient between the abscission and proximal zones, but the distal zone tissue was as active in protein synthesis as the abscission zone. Differences in uptake of the leucine were also observed between different zones and treatments. Regardless of the tissue or the treatment, there was a sharp increase in uptake at the 24 hour point.Direct incubation of abscission zones in IAA and gibberellic acid (GA) indicated that the action of gibberellic acid on abscission is probably through a stimulation of protein synthesis, while IAA seems to act by maintaining the existing rate of protein synthesis in the cells of the abscission zone.