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.

The production of 4-adminobutyrate in response to treatments reducing cytosolic pH and the regulation of intracellular pH

GABA (4-aminobutyrate) is synthesized through the decarboxylation of LGlu- (L-Glu-+ H+ ---> GABA + C02), and compared to many free amino acids is present in high concentrations in plant cells. GABA levels rise rapidly and dramatically in response to varied stress conditions including anaerobiosis. Recent papers suggest that GABA production and associated H+ consumption are parts of a metabolic pH-stat mechanism which ameliorates the intracellular pH decline associated with anaerobiosis or other treatments. To test this hypothesis GABA production and efflux have been measured in isolated Asparagus sprengeri cells in response to three treatments which potentially cause intracellular acidification. Acid loads were imposed using 60 min of (i) anaerobiosis, (ii) H+/LGlu- cotransport, and (iii) treatment with permeant weak acids (butyric, acetic and propionic). Both intra- and extracellular GABA concentrations increased more than 100% after anaerobiosis, almost 1000% after H+/L-Glu- cotransport (light or dark) and almost 5000/0 after addition of 5 mM butyric acid at pH 5.0. HPLC analysis of amino acids indicates that as GABA concentrations increased in response to butyric acid addition, glutamate concentrations decreased. Time-course studies demonstrated that added butyric acid stimulates GABA production by 2800/0 within 15 seconds. A fluorescent determination of cytosolic pH indicates that addition of butyric or other weak acids resulted in a rapid reduction in cytosolic pH of 0.6 pH units. The half time for the response to butyric acid addition is 2.1 seconds, indicating that the decline in cytosolic pH is rapid enough to account for the rapid stimulation of GABA production. The acid load in response to butyric acid addition was assayed by measurements of 14C-butyric acid uptake. Calculations indicate that GABA production accounted for 45% of the imposed acid load. The biological significance of GABA efflux is not yet understood. The results support the original hypothesis suggesting a role for GABA production in cellular pH regulation.

Trading nitrogen for carbon: Nitrogen and carbon translocation in a plant/fungal (Metarhizium spp.) symbiosis

While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria and the microbial decomposition of organic matter. In the majority of cases, symbiotic microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of all land plants form intimate symbiotic relationships with root colonizing fungi. These common plant/fungal interactions have been defined largely through nutrient exchange, where the plant receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal endophytes are common plant colonizers. A number of these fungal species have a dual life cycle, meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships between various plant species and endophytic, insect pathogenic fungi help to improve overall plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for plant carbon. Overall these studies provide the first evidence of nutrient exchange between an insect pathogenic fungus and plants, a relationship that has potentially useful implications on plant primary production, soil health, and overall ecosystem stability.