Machteld C. Mok

O-Glucosylation of cis-Zeatin in Maize. Characterization of Genes, Enzymes, and Endogenous Cytokinins

trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. The existence of a maize (Zea mays) gene (cisZOG1) encoding an O-glucosyltransferase specific to cis-zeatin (R.C. Martin, M.C. Mok, J.E. Habben, D.W.S. Mok [2001] Proc Natl Acad Sci USA 98: 5922–5926) lends further support to this view. Results described here include the isolation of a second maize cisZOG gene, differential expression ofcisZOG1 and cisZOG2, and identification of substantial amounts of cis-isomers in maize tissues. The open reading frame of cisZOG2 has 98.3% identity to cisZOG1at the nucleotide level and 97.8% at the amino acid level. The upstream regions contain common and unique segments. The recombinant enzymes have similar properties, K m values of 46 and 96 μm, respectively, for cis-zeatin and a pH optimum of 7.5. Other cytokinins, including N6-(Δ2-isopentenyl)adenine, trans-zeatin, benzyladenine, kinetin, and thidiazuron inhibited the reaction. Expression of cisZOG1 was high in maize roots and kernels, whereas cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-zeatin riboside, and theirO-glucosides were detected in all maize tissues, with immature kernels containing very high levels of theO-glucoside of cis-zeatin riboside. The results are a clear indication that O-glucosylation of cis-zeatin is a natural metabolic process in maize. Whether cis-zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated.

A maize cytokinin gene encoding an O-glucosyltransferase specific to cis-zeatin

Zeatin is a naturally occurring cytokinin. Biosynthesis and metabolism studies of zeatin have been directed mostly at the trans isomer, although cis-zeatin and its riboside occur as major components in some plant species. It is not known whether parallel regulatory pathways exist for the two isomers. Based on the sequence of the gene ZOG1 encoding a trans-zeatin O-glucosyltransferase from Phaseolus (EC 2.4.1.203), a cis-zeatin-specific O-glucosyltransferase was isolated from maize. This gene, cisZOG1, contains an ORF of 1,401 nucleotides encoding a protein of 51.1 kDa with 41% identity to the Phaseolus ZOG1 protein. Unexpectedly, the maize enzyme recognizes as substrates cis-zeatin and UDP-glucose but not cis-ribosylzeatin, trans-zeatin, or trans-ribosylzeatin. This finding indicates the existence of cis-specific regulatory elements in plants and suggests that cis-zeatin and derivatives may be more important in cytokinin homeostasis than currently recognized.

Partial Purification of a cis-trans-Isomerase of Zeatin from Immature Seed of Phaseolus vulgaris L

nvestigation of the conversion of exogenous cis-zeatin to trans-zeatin in immature seeds of Phaseolus vulgaris L. led to the isolation of a cis-trans-isomerase from the endosperm. The enzyme was purified more than 2000-fold by chromatography on a series of fast protein liquid chromatography (anion exchange, gel filtration, and hydrophobic interaction) and concanavalin A columns. The enzymic reaction favors conversion from the cis to the trans form and requires flavin, light, and dithiothreitol. cis-Zeatin riboside is also a substrate for the enzyme. Retention on the concanavalin A column indicated that the enzyme is a glycoprotein. The enzyme was stable for at least 8 weeks when stored at -80[deg] C. The occurrence of cis-trans-isomerization suggests that cis-zeatin and cis-zeatin riboside formed by tRNA degradation could be precursors of biologically active cytokinins.

Cytokinins: biosynthesis, metabolism and perception.

SummaryCytokinins are essential hormones for plant growth and development. They are also of vital importance for in vitro manipulations of plant cells and tissues. The biological activities and chemistry of cytokinins are well defined but very little is known about their mode of action and it is only recently that cytokinin genes have been identified in plants. This review summarizes the current status of knowledge on cytokinin biosynthesis, metabolism and signal transduction, with an emphasis on genes encoding metabolic enzymes and putative receptors, and genes rapidly induced by cytokinins.

Over-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation

To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays L.) transformants harbouring the ZOG1 gene (encoding a zeatin O-glucosyltransferase from Phaseolus lunatus L.) under the control of the constitutive ubiquitin (Ubi) promoter were generated. The roots and leaves of the transformants had greatly increased levels of zeatin-O-glucoside. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin-deficient plants, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. Transformant leaves had a higher chlorophyll content and increased levels of active cytokinins compared with those of non-transformed sibs. The Ubi:ZOG1 plants exhibited delayed senescence when grown in the spring/summer. While hemizygous transformants had reduced tassels with fewer spikelets and normal viable pollen, homozygotes had very small tassels and feminized tassel florets, resembling tasselseed phenotypes. Such modifications of the reproductive phase were unexpected and demonstrate a link between cytokinins and sex-specific floral development in monocots.

Interspecific hybridization of phaseolus vulgaris with P. lunatus and P. acutifolius

SummaryThe influence of genotypic combinations on the growth of hybrid embryos between Phaseolus vulgaris and P. lunatus, and between P. vulgaris and P. acutifolius was examined. All embryos obtained from P. vulgaris × P. lunatus crosses developed only to a stage which appears to be comparable to the pre-heart-shape stage of selfed embryos. Reciprocal crosses were attempted, but pods abscised at a very early stage. Embryos derived from P. vulgaris × P. acutifolius and reciprocal crosses attained the cotyledon stage although no mature seeds were formed. A distinct characteristic of these embryos was the uneven development of the two cotyledons. The rate of growth and final size of these hybrid embryos seemed to be influenced by the genotypes of both parents.Immature embryos were cultured on defined medium and the effects of glutamine and gibberellin (GA3) were examined. Glutamine was effective in increasing the survival rate; gibberellin had no apparent effect. Plants derived from cultured embryos of P. vulgaris × P. lunatus, P. vulgaris × P. acutifolius and P. acutifolius × P. vulgaris were obtained.

Topolins and Hydroxylated Thidiazuron Derivatives Are Substrates of Cytokinin O-Glucosyltransferase with Position Specificity Related to Receptor Recognition

Glucosides of trans-zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results.

Micropropagation ofPyrus andCydonia and their responses to Fe-limiting conditions

Appropriate micropropagation regimes were determined for fourPyrus species (P. amygdaliformis Vill.,P. betulaefolia Bunge,P. calleryana Dene., andP. communis L.) andCydonia oblonga L. Shoot multiplication was optimal at 10 or 20μM N6-benzyladenine and high light intensity (135μE m−2 s-1). Root formation of thePyrus species was stimulated by exposure of shoots to high levels (10 or 32μM) ofβ-indolebutyric acid (IBA) for 7 days or a dip in 10 mM IBA for 15 s, followed by a passage on auxin-free medium.α-Naphthaleneacetic acid was more effective than IBA in stimulating rooting ofC. oblonga. The effects of Fe-limiting conditions in vitro were determined by comparing the responses of shoots and rooted plantlets to medium containing FeEDTA or FeSO4, with or without bicarbonate. Symptoms of Fe deficiency were genotype-dependent and most severe in the presence of FeSO4 and bicarbonate. Chlorosis was pronounced inCydonia, absent fromP. amygdaliformis andP. communis, and intermediate inP. betulaefolia andP. calleryana, indicating a good correlation between in vitro and field responses. Similar responses were obtained with rooted and unrooted shoots. These findings suggest that in vitro cultures may be used for studies of Fe-chlorosis as well as screening for tolerant rootstocks.

Fertilization and early embryo development in reciprocal interspecific crosses of Phaseolus.

SummaryFertilization and early embryo and endosperm development were examined in Phaseolus vulgaris x P. acutifolius, P. vulgaris x P. lunatus crosses and their reciprocals. The number and length of pollen tubes were not different between selfings and interspecific crosses. Fertilization was completed in all matings and the time of fertilization was maternally dependent which may reflect the degree of maturation of embryo sacs at pollination. A large difference between reciprocal crosses was found in the time of endosperm and embryo division in relation to the time of fertilization. When P. vulgaris was the female parent and P. acutifolius the male parent, endosperm division occurred at the same time as in P. vulgaris upon selfing, while in P. vulgaris x P. lunatus crosses the time of endosperm division was intermediate as compared with the two parents. The time lapse between fertilization and endosperm and embryo division in P. acutifolius x P. vulgaris crosses was longer than in either parent upon selfing. In P. lunatus x P. vulgaris crosses, endosperm division occurred in only 7–12% of the ovules at 72 hours after pollination. Embryo development in these ovules was limited to the four cell stage although the endosperm was at the free nuclei stage. The severe delay in embryo and endosperm divisions may be the major cause of early pod abscission in P. lunatus x P. vulgaris crosses.

Embryo development in reciprocal crosses of Phaseolus vulgaris L. and P. coccineus Lam.

SummaryEmbryo development was examined in reciprocal crosses of Phaseolus vulgaris cv. ‘Great Northern’ and P. coccineus cv. ‘Scarlet Runner’. The formation of abnormal (shrunken and underdeveloped) embryos constituted the primary crossing barrier between the two species when P. coccineus was the female parent. Plants of P. coccineus X P. vulgaris were obtained by embryo culture. Although the P. vulgaris X P. coccineus cross resulted in normal seed development, the fertility of the resulting hybrids was much lower (27%) than that of the reciprocal hybrids (81%). Three classes of F2 embryos, normal, shrunken, and underdeveloped were formed on reciprocal F1s and the frequencies did not differ between reciprocal populations. Thus, the interactions between embryo and endosperm and/or maternal parent rather than cytoplasmic-nuclear effects seem to be important in the determination of the extent of embryo growth. The examination of pollen fertility of F2 plants and the development of F2 and F3 embryos suggests that the formation of abnormal embryos and reduced male fertility are independent events. The P. vulgaris — P. coccineus crosses may be useful in studying the possible involvement of interspecific differences in hormonal metabolism in the development of hybrid embryos.