Ethylene is involved in the nodulation phenotype of Pisum sativum R50 (sym 16), a pleiotropic mutant that nodulates poorly and has pale green leaves.

R50 is characterized as a pleiotropic pea mutant; it forms few nodules and has short lateral roots, short stature and pale leaves. Using grafting techniques, R50 paleness was found to be controlled by the shoot of the mutant whereas the nodulation phenotype was regulated by its root. The paleness of R50 is due to a lower than normal total chlorophyll content in its young leaves. The defect appears to be overcome with age because, as the plant matures, the chlorophyll levels increase in the older leaves. The reduction in stature is attributed to shorter internodes, and the oldest internodes are thicker than those of the parent Sparkle. Upon rhizobial inoculation, R50 forms as many infection threads as Sparkle. However, most of these are arrested in the inner cortex. The threads appear to have lost their directional growth towards the stele, and they coil around within enlarged cortical cells. In addition, very few infection threads are associated with divisions of the inner cortical cells. These aborted nodule primordia are abnormal, flat and mainly composed of cells which have divided anticlinally only. Nodulation of R50 was restored by treating the roots with ethylene inhibitors. The R50 mutant further supports the postulated role of ethylene in regulating rhizobial infection with a probable role in the control of the primordium development.

The Synthesis of [gamma]-Aminobutyric Acid in Response to Treatments Reducing Cytosolic pH.

[gamma]-Aminobutyric acid (GABA) synthesis (L-glutamic acid + H+ -> GABA + CO2) is rapidly stimulated by a variety of stress conditions including hypoxia. Recent literature suggests that GABA production and concomitant H+ consumption ameliorates the cytosolic acidification associated with hypoxia or other stresses. This proposal was investigated using isolated asparagus (Asparagus sprengeri Regel) mesophyll cells. Cell acidification was promoted using hypoxia, H+/L-glutamic acid symport, and addition of butyrate or other permeant weak acids. Sixty minutes of all three treatments stimulated the levels of both intracellular and extracellular GABA by values ranging from 100 to 1800%. At an external pH of 5.0, addition of 5 mM butyrate stimulated an increase in overall GABA level from 3.86 (0.56 [plus or minus] SE) to 20.4 (2.16 [plus or minus] SE) nmol of GABA/106 cell. Butyrate stimulated GABA levels by 200 to 300% within 15 s, and extracellular GABA was observed after 10 min. The acid load due to butyrate addition was assayed by measuring [14C]butyrate uptake. After 45 s of butyrate treatment, H+-consuming GABA production accounted for 45% of the imposed acid load. The cytosolic location of a fluorescent pH probe was confirmed using fluorescent microscopy. Spectrofluorimetry indicated that butyrate addition reduced cytosolic pH by 0.60 units with a half-time of approximately 2 s. The proposal that GABA synthesis ameliorates cytosolic acidification is supported by the data. The possible roles of H+ and Ca2+ in stimulating GABA synthesis are discussed.

Ethylene Inhibitors Partly Restore Nodulation to Pea Mutant E 107 (brz).

E107 (brz) is a pleiotropic mutant of pea (Pisum sativum L. cv Sparkle) characterized by low nodulation, leaf necrosis, excessive ion accumulation, and decreased plant size. The defective nodulation of E107 was studied by light microscopy of lateral roots. The number of infections per centimeter of lateral root was only a third that of Sparkle. Moreover, most of the infections were aborted early; i.e. in only 14% of the infections did the infection thread penetrate beyond the epidermis. Nodulation of E107 was partly restored by treating the plant with the ethylene inhibitors aminoethoxyvinylglycine (AVG) or Ag(+). Treatment with Ag(+) did not increase the number of infections, but half of the infections went to completion. Ag(+) and AVG did not alter the size of the mutant, the accumulation of cations in its shoots, nor the leaf necrosis. Thus, in E107, nodule development can be uncoupled from other pleiotropic characteristics.

Light Microscopy Study of Nodule Initiation in Pisum sativum L. cv Sparkle and in Its Low-Nodulating Mutant E2 (sym 5).

We compared nodule initiation in lateral roots of Pisum sativum (L.) cv Sparkle and in a low-nodulating mutant E2 (sym 5). In Sparkle, about 25% of the infections terminated in the epidermis, a similar number stopped in the cortex, and 50% resulted in the formation of a nodule meristem or an emerged nodule. The mutant E2 (sym 5) was infected as often as was the parent, and it formed a normal infection thread. In the mutant, cell divisions rarely occurred in advance of the infection thread, and few nodule primordia were produced. Growing the mutant at a low root temperature or adding Ag(+) to the substrate increased the number of cell divisions and nodule primordia. We conclude that, in the E2 line, the infection process is arrested in the cortex, at the stage of initial cell divisions before the establishment of a nodule primordium.

Evaluation of the specificity of lectin binding to sections of plant tissue.

Hand sections of young corn root tips have been used in a study of problems encountered in the binding of fluorescently-labelled lectins to plant tissues. It was found, surprisingly, that with lectins specific for a sugar known to be present (Lotus and Ulex lectins for L-fucose), with a lectin specific for a sugar thought not to be present (wheat-germ agglutinin for N-acetylglucosamine), with non-lectin glycoprotein and protein (gamma-globulin and bovine serum albumin) and with basophilic dyes (alcian blue and toluidine blue), a coincidental binding pattern similar to the pattern of autofluorescence in the same tissue was obtained. Corn root tissues include cell walls composed of complex polysaccharides esterified with ferulic acid residues, as well as mucilages which are highly hydrated and expanded. In such material, neither standard inhibition controls with haptens nor the use of a wide range of lectin concentrations are adequate to distinguish clearly specific and non-specific binding of fluorescently-labelled lectin. Therefore, lectins are not the simple test probes they have been supposed. Before interpreting results obtained in using fluorescently-labelled lectins on any tissue sections, all available information (biochemical as well as histochemical) about the tissue must be considered.