Does Gamma-aminobutyric acid function as a plant resistance mechanism against phytophagous insect activity? /

Gamma-aminobutyric acid (GAB A) is a ubiquitous non-protein amino acid synthesized via the decarboxylation of L-glutamate in a reaction catalyzed by the cytosolic enzyme L-glutamate decarboxylase (GAD). In animals it functions as an inhibitory neurotransmitter. In plants it accumulates rapidly in response to various stresses, but its function remains unclear. The hypothesis that GABA accumulation in leaf tissue may function as a plant resistance mechanism against phytophagous insect activity was investigated. GABA accumulation in response to mechanical stimulation, mechanical damage and insect activity was demonstrated. In wt tobacco (Nicotiana tabacum cv Samsun), mechanical stimulation or damage caused GABA to accumulate within 2 min from mean levels of 14 to 37 and 1~9 nmol g-l fresh weight (FW), respectively. In the transgenic tobacco strain CaMVGAD27c overexpressing Petunia GAD, the same treatments caused GABA to accumulate from 12 to 59 and 279 nmol g-l FW, respectively. In the transgenic tobacco strain CaMVGADilC 11 overexpressing Petunia GAD lacking an autoinhibitory domain, mechanical stimulation or damage caused GABA to accumulate from 180 to 309 and 630 nmol g-l FW, respectively. Ambulatory activity by tobacco budworm (TBW) larvae (Heliothis virescens) on leaves of CaMVGAD27c tobacco caused GABA to accumulate from 28 to 80 nmol g-l FW within 5 min. Ambulatory and leaf-rolling activity by oblique banded leaf roller (OBLR) larvae (Choristoneura rosaceana cv Harris) on wt soybean leaves (Glycine max cv Harovinton) caused GABA to accumulate from 60 to 1123 nmol g-l FW within 20 min. Increased GABA levels in leaf tissue were shown to affect phytophagous preference in TBW larvae presented with wt and transgenic tobacco leaves. When presented with leaves of Samsun wt and CaMVGAD27c plants, TBW larvae consumed more wt leaf tissue (640 ± 501 S.D. mm2 ) than transgenic leaf tissue (278 ± 338 S.D. mm2 ) nine times out of ten. When presented with leaves of Samsun wt and CaMVGAD~C11 plants, TBW larvae consumed more transgenic leaf tissue (1219 ± 1009 S.D. mm2 ) than wt leaf tissue (28 ± 31 S.D. mm2 ) ten times out of ten. These results indicate that: (1) ambulatory activity of insect larvae on leaves results in increased GABA levels, (2) transgenic tobacco leaves with increased capacity for GABA synthesis deter feeding, and (3) transgenic tobacco leaves with constitutively higher GABA levels stimulate feeding.

Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.

An examination of transcriptional and translational regulation of the 70kDa heat shock proteins following amputation of the tail and forelimb in the newt Notophthalmus viridescens

Several stresses to tissues including hyperthermia, ischemia, mechanical trauma and heavy metals have been demonstrated to affect the regulation of a subset of the family of heat shock proteins of70kOa (hsp70). In several organisms following some of these traumas, the levels of hsp70 mRNA and proteins are dramatically upregulated. However, the effects of the stress on limb and tail amputation in the newt Notophthalmus viridescens, involving mechanical tissue damage, have not adequately been examined. In the present study, three techniques were utilized to quantitate the levels of hsp70 mRNA and protein in the tissues of the forelimbs and tails of newts during the early post-traumatic events following surgical resection of these:: appendages. These included quantitative Western blotting of proteins separated by both one and twodimensional SDS-polyacrylamide gel electrophoresis and quantitative Northern blot analysis of total RNA. In tissues of both the limb and tail one hour after amputation, there were no significant differences in the levels of hsp70 protein measured by one-dimensional SOSPAGE followed by Western blotting, when compared to the levels measured in the unamputated limb. A 30 minute heat shock at 35°C failed to elicit an increase in the levels of hsp70 protein in these tissues. Further analysis using the more sensitive 20 PAGE separation of stump tissue proteins revealed that at least some of the five hsp70 isoforms of the newt may be differentially regulated in limbs and tails in response to trauma. It appears also that amputation of the tail and limb tissues leads to slight 3 elevation in the levels of HSP70 mRNA when compared to those of their respective unstressed tissues.

Active isolated stretching : an investigation of the mechanical mechanisms

The Active Isolated Stretching (AIS) technique proposes that by contracting a muscle (agonist) the opposite muscle (antagonist) will relax through reciprocal inhibition and lengthen without increasing muscle tension (Mattes, 2000). The clinical effectiveness of AIS has been reported but its mechanism of action has not been investigated at the tissue level. Proposed mechanisms for increased range of motion (ROM) include mechanical or neural changes, or an increased stretch tolerance. The purpose of the study was to investigate changes in mechanical properties, i.e. stiffness, of skeletal muscle in response to acute and long-term AIS stretching for the hamstring muscle group. Recreationally active university-aged students (female n=8, male n=2) classified as having tight hamstrings, by a knee extension test, volunteered for the study. All stretch procedures were performed on the right leg, with the left leg serving as a control. Each subject was assessed twice: at an initial session and after completing a 6-week AIS hamstring stretch training program. For both test sessions active knee extension (ROM) to a position of "light irritation", passive resisted torque and stiffness were determined before and after completion of the AIS technique (2x10 reps). Data were collected using a Biodex System 3 Pro (Biodex Medical Systems, NY, USA) isokinetic dynamometer. Surface electromyography (EMG) was used to monitor vastus lateralis (VL) and hamstring muscle activity during the stretching movements. Between test sessions, 2x10 reps of the AIS bent knee hamstring stretch were performed daily for 6-weeks.

The Influence of Sex on the Relationship Between Arterial Mechanical Properties and Cardiovagal Baroreflex Sensitivity

Cardiovagal baroreflex sensitivity (cvBRS) demonstrates a strong relationship with arterial mechanical properties. Both cvBRS and arterial mechanics differ by sex such that males demonstrate greater cvBRS, yet lower large artery elasticity than females. Whether the relationship between cvBRS and arterial mechanics is similar in males and females remains unexamined. As a result, it is unclear whether arterial mechanics contribute to sex differences in cvBRS. This study investigated the cross-sectional relationship between cvBRS and arterial mechanical properties of the common carotid, carotid sinus and aortic arch (AA) in 36 (18 females) young, healthy normotensives. The cvBRS-arterial mechanics relationship did not reach statistical significance and did not differ by sex. Both cvBRS and AA distensibility were greater in females than males. Sex differences in cvBRS were eliminated after controlling for AA distensibility. These findings suggest that in this sample, AA elasticity may contribute to the greater cvBRS in females than males.