Effect of Auxins (2,4-Dichlorophenoxyacetic acid, Indole-3-Acetic Acid and Naphthaleneacetic Acid) on the Accumulation of {gamma}-Aminobutyric Acid in Excised Rice Root Tips

-Aminobutyric acid (GABA) accumulation occurs in cultured ricecells when ammonium is added to the medium [Kishinami and Ojima(1980) Plant Cell Physiol. 21: 581–589]. Whether thisphenomenon occurs in rice plant tissues was examined with respectto exogenously supplied auxins: 2,4-dichlorophenoxyacetic acid(2,4-D), indole-3-acetic acid (IAA) and naphthalene-acetic acid(NAA). In intact rice plants grown in medium containing ammonium withoutauxin, glutamine first increased, then asparagine graduallyincreased. In both shoots and roots, the asparagine contentbecame the highest among four amino acids after 4 days of cultureperiod. GABA did not increase at all, its level remaining lowin both shoots and roots throughout the culture period. GABA accumulation was observed in excised rice root tips whenthey were incubated in the medium containing ammonium in thepresence of 2,4-D, IAA or NAA. In the absence of auxin, however,excised rice root tips accumulated asparagine and glutamine,but not GABA. Rice root segments obtained from a region in whichroot cells had already developed to maturity did not accumulateGABA but asparagine and glutamine in the presence of both ammoniumand 2,4-D. These results suggest that GABA accumulation occurs in rapidlygrowing and dividing tissue, such as the apical meristem ofrice root in the presence of auxin during ammonium assimilation. (Received June 15, 1987; Accepted March 14, 1988)     

Allyl Alcohol Selection for Lower Alcohol Dehydrogenase Activity in Nicotiana plumbaginifolia Cultured Cells

One cell strain with stable tolerance to allyl alcohol (AA^r) was selected from 6 × 10⁸ suspension cultured Nicotiana plumbaginifolia Viviani cells. The selected strain contained one-half the alcohol dehydrogenase (ADH) activity of the wild type (NP) due to the loss of two of three bands of ADH activity seen on starch gels following electrophoresis of wild-type cell extracts. Anaerobic conditions, simulated by not shaking the suspension cultures, increased the ADH specific activity to more than 3-fold the initial level in both strains but did not change the number of activity bands or the relative levels of activity. The cell strain with decreased ADH activity lost viability more rapidly than the wild type under the anaerobic conditions. The AA^r cells were 10 times more tolerant to ethanol than the NP cells and were also somewhat more tolerant to acetaldehyde and antimycin A. The substrate specificities of the ADH enzymes from both strains were very similar. Further selection of AA^r cells with allyl alcohol produced strains with even lower ADH activity and selection under anaerobic conditions produced strains with increased ADH activity. Genetic studies indicate that the N. plumbaginifolia ADH activity bands arise from subunits produced by two nonallelic genes. This is the first example of the use of allyl alcohol to select for decreased ADH using cultured plant cells.     

Characterization of Cu and Zn Resistant Nicotiana plumbaginifolia Suspension Cultures

The mechanisms of Cu and Zn resistance in cultured Nicotianaplumbaginifolia cells were investigated. When crude extractsof both the Cu and Zn resistant cells were passed through SephadexG-75 gel filtration columns, two metal containing peaks wereidentified. In each case the Cu or Zn was associated with ahigh molecular weight fraction (ca. 70,000) and a low molecularweight fraction (less than 3,000) with the majority being associatedwith the low molecular weight fraction. The low molecular weightfraction in particular increased upon exposure of the cellsto the metals. Both the Cu and Zn resistant cells accumulated higher levelsof citrate and malate than the wild type cells in the presenceof the metals. The addition of citrate or malate, but not fumarateor succinate to the culture medium prevented Cu or Zn toxicityto wild type cells. These results suggest that the N. plumbaginifoliacells selected as resistant to Cu or Zn are resistant, at leastin part, because of their ability to accumulate the chelators,citrate and malate, in the presence of the metals. ¹Present address: Plantech Research Institute, MCI ResearchCenter, 1000 Kamoshida-cho, Midoriku, Yokohama 227 Japan (Received June 30, 1986; Accepted December 1, 1986)     

Auxotrophic Complementation in Intergeneric Hybrid Cells Obtained by Electrical and Dextran-Induced Protoplast Fusion

The auxotrophic mutants, NX1 (Nicotiana plumbagintfolia Viviani,lacking nitrate reductase) and 8B9 (Hyoscyamus muticus, tryptophanrequiring) were used in protoplast fusion experiments usingboth electrical stimulation and dextran as fusion agents. Inboth cases growing colonies were recovered when the fused protoplastswere plated directly in minimal medium lacking tryptophan withnitrate as the nitrogen source. The parental cells did not surviveon this medium. A cold pretreatment for 1 h at 4?C was necessaryto recover hybrids after electrical fusion. The putative hybridscontinued to grow on minimal medium, contained nitrate reductaseactivity and had alcohol dehydrogenase and esterase isozymepatterns at least in part in common with those of the parents.Two new esterase bands were expressed in the hybrids. Thesestudies show that both electrical and dextran induced protoplastfusion can produce hybrids selectable by auxotrophic complementation. ¹Present address: Plantech Research Institute, 1000 Kamoshidacho,Midoriku, Yokohama, Kanagawa 227, Japan (Received September 1, 1986; Accepted December 2, 1986)     

Accumulation of {gamma}-Aminobutyric Acid due to Ammonium in Various Cultured Plant Cells

The accumulation of -aminobutyric acid (GABA) resulting fromthe addition of ammonium was investigated with various culturedplant cells including carrot, corn, peanut, rice, rue, soybean,tobacco and wheat. All rice cell lines tested (10 japonica,12 indica types and 3 Oryza species other than 0. saliva) showedmarked accumulation of GABA in the presence of ammonium in themedium. This accumulation was observed in not only seed-derivedrice cells but also anther-derived rice cells. But culturedcells other than rice did not accumulate GABA. Alanine (in cornand wheat) and glutamine in carrot, peanut, rue, soybean andtobacco) were the most abundant free amino acids in those cellsin the presence of ammonium. These results indicate that theaccumulation of GABA due to the addition of ammonium occursspecifically in rice cells and not in the cells of genera otherthan Oryza thus far investigated. (Received January 24, 1987; Accepted September 1, 1987)     

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv Igri) were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.     

Accumulation of {gamma}-aminobutyric acid due to adding ammonium or glutamine to cultured rice cells

-Aminobutyric acid (GABA) was accumulated in rice cell cultureswhen ammonium was added to the medium. After the addition ofammonium, a temporary increase in the glutamine (Gln) pool wasobserved before the accumulation of GABA. GABA also was markedlyaccumulated when Gln was added to the medium in place of ammonium.When glutamic acid (Glu) was added without ammonium, no accumulationof GABA occurred. When L-methionine-DL-sulfoximine (MSO), an inhibitor of glutaminesynthetase, was added to the nitrate medium, the ammonium poolincreased with no accumulation of Gln and GABA. Even when ammoniumwas supplied to the medium, no GABA accumulated in the presenceof MSO. When azaserine (AZ), which inhibits the transamidationof Gln, was added with Gln, no GABA was accumulated, althoughthe Gln pool in the cell cultures increased significantly. The accumulation of GABA in cultured rice cells produced byammonium as the nitrogen source probably is related directlyto the Gln pool size, which is increased when ammonium is suppliedto the medium. (Received August 6, 1979; )     

Selection of Copper and Zinc Resistant Nicotiana plumbaginifolia Cell Suspension Cultures

Copper and zinc resistant cells of Nicotiana plumbaginifoliawere selected using unmutagenized cell suspensions in mediumcontaining normally lethal concentrations of CuSO₄ or ZnSO₄.Both resistances were retained for thirty cell doublings withoutselection pressure. The Cu resistant cells were 10-times andthe Zn resistant cells were 6-times as resistant as the wildtype cells. The Zn resistant cells were also somewhat resistantto AlCl₃ in comparison with the wild type cells, while the Curesistant cells were also somewhat resistant to ZnSO₄ and AlCl₃.The uptake of Cu by the Cu resistant cells and Zn by the Znresistant cells was higher than that of the wild type cells. (Received April 21, 1986; Accepted June 30, 1986)