Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses—nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

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Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved

Trans -isomers of cytokinins (CK) are thought to predominate and have greater biological activity than corresponding cis -isomers in higher plants. However, this study demonstrates a system within which the predominant CK are cis -isomers. CK were measured at four developmental stages in developing chickpea ( Cicer arietinum L. cultivar Kaniva) seeds by gas chromatography-mass spectrometry. Concentrations were highest at an early endospermic fluid stage and fell considerably when the cotyledons expanded. The cis -isomers of zeatin nucleotide ([9R-MP]Z), zeatin riboside ([9R]Z), and zeatin (Z) were present in greater concentrations than those of corresponding trans -isomers: ( trans )[9R-MP]Z, ( trans )[9R]Z, ( trans )Z, or dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and the isopentenyl-type CK concentrations were either low or not detectable. Root xylem exudates also contained predominantly cis -isomers of [9R-MP]Z and [9R]Z. Identities of ( cis )[9R]Z and ( cis )Z were confirmed by comparison of ion ratios and retention indices, and a full spectrum was obtained for ( cis )[9R]Z. Tissues were extracted under conditions that minimized the possibility of RNase hydrolysis of tRNA following tissue disruption, being a significant source of the cis -CK. Since no isomerization of ( trans )[ 2 H]CK internal standards occurred, it is unlikely that the cis -CK resulted from enzymic or nonenzymic isomerization during extraction. Although quantities of total CK varied, similar CK profiles were found among three different chickpea cultivars and between adequately watered and water-stressed plants. Developing chickpea seeds will be a useful system for investigating the activity of cis -CK or determining the origin and metabolism of free CK.

cis-Isomers of Cytokinins Predominate in Chickpea Seeds throughout Their Development

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified The N 6 -side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation

Cytokinin biosynthesis and interconversion

The role of plant hormones in aluminium (Al3+)-inducedinhibition of root growth was investigated in roots of Phaseolusvulgaris L. cv. Strike. Changes in ethylene evolution and changes inthe content and composition of cytokinins (CKs), at intervals up to 150min after treatment with Al3+, were determined by gaschromatography (GC) and combined gas chromatography-mass spectrometry (GC-MS),respectively. Seedlings were cultivated in a continuously aerated nutrientsolution at pH 4.5 with and without Al3+. The growth rate ofAl3+-treated roots was significantly decreased after 360min. Ethylene evolution from excised root tips doubled after 15min of Al3+ treatment and reached a maximum 30min after treatment. Levels of CK nucleotides declined 60 to 80%after only 5 min of Al3+ treatment whereas the zeatin(Z) content increased six-fold. The increase in Z continued over the entire 150min-sampling period and reached a level 80 times higher than thatin roots not exposed to Al3+. These results show thatAl3+-induced inhibition of root growth is preceded by significantchanges in CK content and composition and enhanced ethylene evolution. Since CKcan induce ethylene production, the rapid increase in CK, particularly Z anddihydrozeatin (dZ), may contribute to inhibition of root-growth either directlyor indirectly by affecting plant hormone homeostasis.

A rapid increase in cytokinin levels and enhanced ethylene evolution precede Al3+-induced inhibition of root growth in bean seedlings (Phaseolus vulgaris L.)

Correlative data from monocots suggest that cytokinin (CK) regulates seed development. The involvement of CKs in seed growth was investigated using pea, a eudicot with an unknown CK profile, as a model system. CK profiles were measured by liquid chromatography – tandem mass spectrometry against major stages of embryogenesis, which were documented histologically. Like other grain legumes, CK levels of developing pea seeds fluctuated through development and had mainly nucleotide and riboside forms. Among the 11 CKs detected, cis-isomers (cis-[9R]Z (zeatin riboside), and cis-[9RMP]Z (zeatin riboside 5′ monophosphate)), along with their isopentenyl precursors, were the major forms during pea embryogenesis, whereas corresponding trans-isomers appeared as minor constituents. Highest CK concentrations occurred at the heart-shape stage, when there are high rates of cell division and sugar metabolism. To assess the significance of high CK concentrations observed at the heart-shape stage, a bioassay was developed w...

cis-Cytokinins that predominate in Pisum sativum during early embryogenesis will accelerate embryo growth in vitro

The determination of the various endogenous cytokinins and their distribution among organs is important in understanding their role in growth and development in the intact plant Cytokinins in young plants of Phaseolus vulgaris were purified by immunoaffinity chromatography and HPLC, and characterised by UV spectra Zeatin nucleotide (zeatin riboside-5'-monophosphate) and isopentenyladenine nucleotide (isopentenyladenosine-5'-monophosphate) were the most abundant cytokinins in all organs Their identities were confirmed by GC-MS The levels of zeatin, zeatin riboside, isopentenyladenine and isopentenyladenosine never exceeded 5% of the nucleotides, as assessed by a methodology that preserves cytokinin nucleotides Three extraction methods were compared with qualitatively similar results, though differing in their suppression of nucleotidase activity Cytokinin nucleotide levels were greater in the stems and petioles than in the roots and leaves on a per gram fresh weight basis, and were greater in the stems than in the other organs on a per plant basis Levels of the zeatin and isopentenyladenine nucleotides were about equal in the stems and leaves, but in the petioles the zeatin nucleotide levels were about twice the level of isopentenyladenine nucleotide, while in the roots they were about half the isopentenyladenine nucleotide level The importance of considering the cytokinin form is emphasised

Ejörn Nicander

Papers

Identification of some major cytokinins in Phaseolus vulgaris and their distribution