Fruits of tomato, Lycopersicon esculentum Mill. cv Liberty, ripen slowly and have a prolonged keeping quality. Ethylene production and the levels of polyamines in pericarp of cv Liberty, Pik Red, and Rutgers were measured in relation to fruit development. Depending on the stage of fruit development, Liberty produced between 16 and 38% of the ethylene produced by Pik Red and Rutgers. The polyamines putrescine, spermidine, and spermine were present in all cultivars. Cadaverine was detected only in Rutgers. Levels of putrescine and spermidine declined between the immature and mature green stages of development and prior to the onset of climacteric ethylene production. In Pik Red and Rutgers, the decline persisted, whereas in Liberty, the putrescine level increased during ripening. Ripe pericarp of Liberty contained about three and six times more free (unconjugated) polyamines than Pik Red and Rutgers, respectively. No pronounced changes in spermidine or cadaverine occurred during ripening. The increase in the free polyamine level in ripe pericarp of Liberty may account for the reduction of climacteric ethylene production, and prolonged storage life.

The exine was isolated from lily pollen which had been treated with 4-methylmorpholine N-oxide and shown by cross-polarization/magic-angle-spinning 13C NMR to be predominantly an aliphatic polymer.

Analyses of the IAA-overproducing mutant of Lemna have been initiated in order to study in vivo biosynthesis of IAA. Using radiolabelled tryptophan isomers prepared from commercial sources of {sup 14}C-D,L tryptophan by chiral separation kinetics of uptake of L and D tryptophan were determined for sterile cultures of individual jsR{sub 1} four-frond colonies. Over a 24 h period, about 50% of the radioactivity from {sup 14}C-L-TRP in media, or about 25% from {sup 14}C-D-TRP, was found in the plant tissue. Maximal rates of uptake were seen in the first six hors for both isomers. Endogenous levels of tryptophan determined in jsR{sub 1} as measures of pool sizes in vivo show 5 to 10 ug/g FW total tryptophan with less than 1% in the D isomer form. Information on uptake and endogenous pool sizes of tryptophan isomers is being used for feeding of stable isotope labeled tryptophan ({sup 13}C, {sup 14}N) to jsR{sub 1} at physiological levels. Analyses of incorporation of label into IAA using GC-MS and high resolution mass spectrometry are currently underway.

Ester conjugates ofindole-3-acetic acidarehydrolyzed easily inbasicsolutions; however, quantitative datahavenotbeen available on therelationship betweenpHandrateofhydrolysis oftheknownester conjugates. Theuseofbasic conditions during extraction orpurification ofIAAbyseveral laboratories suggested that a more systematic analysis ofthis processwas needed. In thisreport we present dataindicating: (a)thatmeasurable hy- drolysis ofIAA-glucose (from standard solutions) andIAA-esters (frommaizekernel extracts) occurswithonlya fewhoursof treatment atpH9 orabove; (b)thatthelability ofsome ester conjugates isevengreater thanthatofIAA-glucose; and(c)that esterhydrolysis ofstandard compounds, IAA-glucose andIAA- p-nitrophenol, occursinthe'three phaseextraction system' pro- posedbyLiuandTillberg ((1983) Physiol Plant57:441-447). Thesedataindicate that thepotential forproblems withinadvert- enthydrolysis ofester conjugates ofIAAexists evenatmoderate pHvalues andinthemultiphase systemwhereexposuretobasic conditions was thought tobelimited. appropriate precautions beemployed toprevent their inad- vertent hydrolysis during extraction andpurification. The mostdetailed studies ofthehydrolysis ofknownIAAesters havecentered ontheconditions necessary forquantitative hydrolysis without appreciable hydrolysis ofamideforms (4, 6)andfewstudies haveanalyzed therelationship between pH andrate ofhydrolysis oftheknownester conjugates. Useof basic conditions during extraction orpurifications ofIAAby several laboratories (see, forexample, refs. 10,11,16,18,22, 23)following various protocols suggested that amoresystem- atic evaluation ofthis process wasneeded. Inthis report we present dataonthekinetics ofhydrolysis ofreagent IAA- GLU2atselected pHvalues andcompare thelability ofIAA- GLUtothat ofIAA-MIandseveral other ester conjugates of IAA.Inaddition, wereport thebehavior ofarelatively polar conjugate, IAA-GLU, andarelatively apolar conjugate, IAA- PNP,inthe'three phase' system described byLiuandTillberg (16). Thesedataindicate thatproblems withinadvertent hydrolysis areapparent evenatmoderate pHvalues andin multiphase extraction systems.

Studies on the subcellular localization and selected properties of consitutive phosphohydrolases in pollen from Lilium longiflorum Thunb. (pH 5 phytase, pH 8 phytase, myo-inositol monophosphatase) are described. In anthers, all three enzymes increased in activity during pollen maturation along with levels of phytic acid. These enzymes were localized in mature pollen by the use of lead capture cytochemistry and shown to be associated with the membrane of the organelle previously identified as the storage site of phytic acid in the form of its insoluble salt, phytin. Recovery of the phytase with optimal activity at pH 8 was most complete when detergent or phospholipase C was included in the extracting medium, suggesting that its association with the organelle membrane is more integral than that of the other enzymes. The pH 8 phytase exhibited differences from a pH 5 phytase which has not been previously reported. They include higher substrate specificity for phytate, terminal hydrolysis to myo-inositol trisphosphate (stereochemistry undetermined), and lack of inhibition of fluoride. Our results suggest a subcellular organization of phytic acid metabolism in lily pollen with the pH 8 phytase as an important component.

Transmission electron microscopy of pollen from Lilium longiflorum Thunb. reveals electron-dense inclusions in storage body organelles ubiquitous in the cytosol. In ungerminated pollen, these inclusions are rounded in appearance and appressed to the inner surface of the smooth membrane of the storage body. During pollen germination, these inclusions become less rounded, smaller, and enclosed in storage bodies that have developed crenated membranes. Energy dispersive x-ray analysis reveals high levels of P, Mg, K, and Ca in the inclusions relative to other regions of the cytosol in which elemental signals can be obtained. The elemental composition and the degradation of inclusions during germination are offered as evidence for storage of phytin in these structures which are thus analogous to phytin storage globoids of seed tissues.

Methods for the removal of exine from mature, ungerminatedLilium longiflorum pollen and release of intact gametophytes (sporoplasts) have been developed. These methods rely on the low temperature solvolytic activity of 4-methylmorpholine N-oxide (MMNO), which allows partial or complete detachment of exine from intine during subsequent washing procedures. These methods are: aqueous MMNO combined with cyclohexylamine (method I), aqueous MMNO at alkaline pH (method II), and aqueous MMNO containing a high Ca2+ concentration with added cellulysin and macerase (method III). Sporoplasts produced by methods I and II are most frequently completely separated from exine and, as shown by histochemical tests, enveloped by the intine layer. Selected enzyme activities in method II sporoplasts are measurable but, as indicated by other tests, considerable damage to the plasma membrane accompanies this treatment. Sporoplasts produced by melhod III largely remain attached to their ruptured exine layer and retain substantial biological competence in terms of extractable enzyme activities, membrane integrity, and respiration.

Methods for preparation of plant protoplasts are well established but the extension of such methods to the release of exine-free gametophytes from pollen appears to be limited (Bajaj 1974; Bajaj and Davey 1974; Bhojwani and Cocking 1972; Power 1973; Takegami and Ito 1975; Zhu et al. 1984). We have discovered that 4-methylmorpholine N-oxide monohydrate (MMNO·H2O) is an effective solvent of the intine layer when pollen grains of Lilium longiflorum Thunb. (trumpet lily) are dispersed in MMNO·H2O at its melting point, 75°C (Loewus et al. 1985). Exine-free gametophytes, which we term sporoplasts, are quickly released from their exine enclosures. With time, MMNO also disperses the empty exine ‘shells’ into immiscible droplets. Prolonged heating ruptures the sporoplasts to produce empty sporoplast envelopes or ‘ghosts’ which remain intact in the MMNO·H2O melt.

A radiochemical synthesis is described for [14C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [14C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles.

4-Methylmorpholine N-oxide monohydrate (MMNO.H(2)O), a potent solvent for polysaccharides, is an effective vehicle for release of membrane-enclosed male gametophytes (sporoplasts) from spore walls. This release occurs in minutes when pollen (Lilium longiflorum Thunb.) is suspended in a melt of MMNO.H(2)O at 75 degrees C. Continued heating at 75 degrees C leads to distintegration of the exine ;shell' which coalesces into immiscible globules in the MMNO melt. These observations provide a general procedure for preparation of pollen sporoplasts and sporoplast outer membranes, and offer a new method for dissolving the sporopollenin component of the spore wall.