Ruth C. Martin

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.

microRNA, seeds, and Darwin?: diverse function of miRNA in seed biology and plant responses to stress

microRNAs (miRNAs) are small, single-stranded RNAs that down-regulate target genes at the post-transcriptional level. miRNAs regulate target genes by guiding mRNA cleavage or by repressing translation. miRNAs play crucial roles in a broad range of developmental processes in plants. Multiple miRNAs are present in germinating seeds and seedlings of Arabidopsis, some of which are involved in the regulation of germination and seedling growth by plant hormones such as abscisic acid (ABA) and auxin. The involvement of miRNAs in ABA responses is not limited to the early stages of plant development but seems to be important for general stress responses throughout the plant life cycle. This Darwin review summarizes recent progress in miRNA research focusing on seed and stress biology, two topics which were of interest to Charles Darwin.

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.

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.

The microRNA156 and microRNA172 gene regulation cascades at post-germinative stages in Arabidopsis

MicroRNAs (miRNAs) are involved in developmental programmes of plants, including seed germination and post-germination. Here, we provide evidence that two different miRNA pathways, miR156 and miR172, interact during the post-germination stages in Arabidopsis. Mutant seedlings expressing miR156-resistant SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE13 (mSPL13), which has silent mutations in the miR156 complementary sequence, over-accumulated SPL13 mRNA and exhibited a delay in seedling development. Microarray analysis indicated that SCHNARCHZAPFEN (SNZ), an AP2-like gene targeted by miR172, was downregulated in these mutants. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and miRNA gel blot analyses showed that the MIR172 genes were up-regulated in mSPL13 mutants. These results suggest that the miRNA regulation cascades (miR156 s SPL13 ! miR172 s SNZ) play a critical role during the post-germination developmental stages in Arabidopsis.

The regulation of post-germinative transition from the cotyledon- to vegetative-leaf stages by microRNA-targeted SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 in Arabidopsis

Germination and early seedling development are critical for successful stand establishment of plants. Following germination, the cotyledons, which are derived from embryonic tissue, emerge from the seed. Arabidopsis seedlings at post-germinative stages are supported mainly by the supply of nutrition from the cotyledons until vegetative leaves emerge and initiate photosynthesis. The switch to autotrophic growth is a significant transition at the post-germinative stage. Here, we provide evidence that down-regulation of SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 (SPL13) by microRNA156 (miR156) plays an important role in the regulation of the post-germinative switch from the cotyledon stage to the vegetative-leaf stage. Silent mutations created in the SPL13 sequence in the region that is complementary to the miR156 sequence caused the deregulation of the mutant form of SPL13 (mSPL13) mRNA from miR156. Mutant seedlings over-accumulated miRNA-resistant messages and exhibited a delay in the emergence of vegetative leaves compared to wild-type seedlings. The delay was not observed in control transgenic plants expressing nonmutated SPL13, indicating that the phenotype was caused specifically by the silent mutations and deregulation of SPL13 from miR156. Characterization of the SPL13 promoter indicated that this gene is expressed mainly in the hypocotyl and affects leaf primordium development. These results suggest that the repression of SPL13 by miR156 is essential for normal post-germinative growth in Arabidopsis.

Seed Traits and Genes Important for Translational Biology—Highlights from Recent Discoveries

Seeds provide food, feed, fiber and fuel. They are also an important delivery system of genetic information, which is essential for the survival of wild species in ecosystems and the production of agricultural crops. In this review, seed traits and genes that are potentially important for agricultural applications are discussed. Over the long period of crop domestication, seed traits have been modified through intentional or unintentional selections. While most selections have led to seed traits favorable for agricultural consumption, such as larger seeds with higher nutritional value than the wild type, other manipulations in modern breeding sometimes led to negative traits, such as vivipary, precocious germination on the maternal plant or reduced seed vigor, as a side effect during the improvement of other characteristics. Greater effort is needed to overcome these problems that have emerged as a consequence of crop improvement. Seed biology researchers have characterized the function of many genes in the last decade, including those associated with seed domestication, which may be useful in addressing critical issues in modern agriculture, such as the prevention of vivipary and seed shattering or the enhancement of yields. Recent discoveries in seed biology research are highlighted in this review, with an emphasis on their potential for translational biology.

Identification and characterization of a salt stress-inducible zinc finger protein from Festuca arundinacea

BackgroundIncreased biotic and abiotic plant stresses due to climate change together with an expected global human population of over 9 billion by 2050 intensifies the demand for agricultural production on marginal lands. Soil salinity is one of the major abiotic stresses responsible for reduced crop productivity worldwide and the salinization of arable land has dramatically increased over the last few decades. Consequently, as land becomes less amenable for conventional agriculture, plants grown on marginal soils will be exposed to higher levels of soil salinity. Forage grasses are a critical component of feed used in livestock production worldwide, with many of these same species of grasses being utilized for lawns, erosion prevention, and recreation. Consequently, it is important to develop a better understanding of salt tolerance in forage and related grass species.FindingsA gene encoding a ZnF protein was identified during the analysis of a salt-stress suppression subtractive hybridization (SSH) expression library from the forage grass species Festuca arundinacea. The expression pattern of FaZnF was compared to that of the well characterized gene for delta 1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in proline biosynthesis, which was also identified in the salt-stress SSH library. The FaZnF and P5CS genes were both up-regulated in response to salt and drought stresses suggesting a role in dehydration stress. FaZnF was also up-regulated in response to heat and wounding, suggesting that it might have a more general function in multiple abiotic stress responses. Additionally, potential downstream targets of FaZnF (a MAPK [Mitogen-Activated Protein Kinase], GST [Glutathione-S-Transferase] and lipoxygenase L2) were found to be up-regulated in calli overexpressing FaZnF when compared to control cell lines.ConclusionsThis work provides evidence that FaZnF is an AN1/A20 zinc finger protein that is involved in the regulation of at least two pathways initiated by the salt stress response, thus furthering our understanding of the mechanisms of cellular action during a stress that is applicable to commercial crops worldwide.

Wounding systemically activates a mitogen-activated protein kinase in forage and turf grasses

Forage and turf grasses are continually cut and grazed by livestock, however very little is known concerning the perception or molecular responses to wounding. Mechanical wounding rapidly activated a 46 kDa and a 44 kDa mitogen-activated protein kinase (MAPK) in six different grass species. In the model grass species Lolium temulentum, the 46 kDa MAPK was rapidly activated within 5 min of wounding both locally and systemically in an adjacent unwounded tiller. This indicates that wounding generates a rapidly propagated long-distance signal that activates a MAPK in the distal portions of the plant. This 46 kDa MAPK activity was not enhanced by the addition of the pathogen-associated signal salicylic acid (SA) to the wound site nor induced when exposed to methyl jasmonate (MJ), which is a potent inducer of the wound response in dicotyledonous plants. However, pretreatment with MJ increased the wound-induced activity of the 44 kDa MAPK over the activity in control plants.