O-linked N-acetylglucosamine (O-GlcNAc) modifications regulate the posttranslational fate of target proteins. The Arabidopsis thaliana O-GlcNAc transferase (OGT) SPINDLY (SPY) suppresses gibberellin signaling and promotes cytokinin (CK) responses by unknown mechanisms. Here, we present evidence that two closely related class I TCP transcription factors, TCP14 and TCP15, act with SPY to promote CK responses. TCP14 and TCP15 interacted with SPY in yeast two-hybrid and in vitro pulldown assays and were O-GlcNAc modified in Escherichia coli by the Arabidopsis OGT, SECRET AGENT. Overexpression of TCP14 severely affected plant development in a SPY-dependent manner and stimulated typical CK morphological responses, as well as the expression of the CK-regulated gene RESPONSE REGULATOR5. TCP14 also promoted the transcriptional activity of the CK-induced mitotic factor CYCLIN B1;2. Whereas TCP14-overexpressing plants were hypersensitive to CK, spy and tcp14 tcp15 double mutant leaves and flowers were hyposensitive to the hormone. Reducing CK levels by overexpressing CK OXIDASE/DEHYDROGENASE3 suppressed the TCP14 overexpression phenotypes, and this suppression was reversed when the plants were treated with exogenous CK. Taken together, we suggest that responses of leaves and flowers to CK are mediated by SPY-dependent TCP14 and TCP15 activities.

A tomato (Lycopersicon esculentum) gene (GAST1) that encodes an RNA whose abundance increases > 20-fold in shoots of the GA-deficient gib1 mutant following spraying with GA3 has been characterized. An increase in GAST1 RNA levels is detectable 2 h after treatment and levels continue to increase for at least an additional 10 h. Between 12 and 24 h following treatment, the amount of GAST1 RNA begins to decline and at 48 h the level is nearly equivalent to that of water-treated control plants. Nuclear runoff analysis indicates that 8 h after treatment with GA3, transcription of the GAST1 gene has increased only threefold, suggesting that GA acts both transcriptionally and post-transcriptionally. ABA partially inhibits the GA-mediated increase in GAST1 RNA abundance while ethephon, kinetin, and 2,4-D have little effect. GAST1 RNA is detectable in untreated leaves, stems, petioles and flowers, but not in roots. The GAST1 gene encodes a 0.7 kb transcript. The sequence of the GAST1 cDNA and genomic clones indicates that the gene is interrupted by three introns and potentially encodes a 112 amino acid protein of unknown function.

SPY (SPINDLY) encodes a putative O-linked N-acetyl-glucosamine transferase that is genetically defined as a negatively acting component of the gibberellin (GA) signal transduction pathway. Analysis of Arabidopsis plants containing a SPY::GUS reporter gene reveals that SPY is expressed throughout the life of the plant and in most plant organs examined. In addition to being expressed in all organs where phenotypes due to spy mutations have been reported, SPY::GUS is expressed in the root. Examination of the roots of wild-type, spy, and gai plants revealed phenotypes indicating that SPY and GAI play a role in root development. A second SPY::GUS reporter gene lacking part of the SPY promoter was inactive, suggesting that sequences in the first exon and/or intron are required for detectable expression. Using both subcellular fractionation and visualization of a SPY-green fluorescent protein fusion protein that is able to rescue the spy mutant phenotype, the majority of SPY protein was shown to be present in the nucleus. This result is consistent with the nuclear localization of other components of the GA response pathway and suggests that SPY's role as a negative regulator of GA signaling involves interaction with other nuclear proteins and/or O-N-acetyl-glucosamine modification of these proteins.GAs are endogenous plant growth regulators that have been studied for over 70 years. Until relatively recently, most of this research has concentrated on determining the physiological role of various GAs, defining the GA biosynthetic pathway in plants and fungi, and developing practical uses for GAs and chemical inhibitors of GA biosynthesis in agriculture. Over the last decade, considerable progress has also been made in understanding how plants are able to perceive the level of endogenous GAs and the mechanism by which the GA signal is transduced (Thornton et al., 1999a;Lovegrove and Hooley, 2000;Sun, 2000; Richards et al., 2001). This research has been made possible by advances in molecular genetic techniques in model systems such as Arabidopsis, rice (Oryza sativa), and the aleurone layer of cereal grains. In Arabidopsis, several negatively acting components of the GA response pathway have been characterized in some detail, including SPY (SPINDLY; Jacobsen and Olszewski, 1993;Jacobsen et al., 1996), and two members of the GRAS family (Pysh et al., 1999), RGA (REPRESSOR OF ga1-3) and GAI (GA INSENSITIVE; Peng et al., 1997; Silverstone et al., 1998). The cloning of GAI has led to the identification of orthologous genes from other species such as the wheat (Triticum aestivum) rht homeo-alleles that are the genetic basis of the "green revolution" (Peng et al., 1999a). Other potential GA-signaling proteins include SHI (SHORT INTERNODES), SLY (SLEEPY), and PKL (PICKLE) in Arabidopsis (Steber et al., 1998; Fridborg et al., 1999;Ogas et al., 1999), and GAMyb in barley (Hordeum vulgare;Gubler et al., 1999). A role for heterotrimeric G proteins has also been suggested based on work with inhibitors in wild oat (Avena sativa) a...

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