Brandon d. Moore

Sugar sensing and signaling in plants

In addition to their essential roles as substrates in carbon and energy metabolism and in polymer biosynthesis, sugars have important hormone-like functions as primary messengers in signal transduction. The pivotal role of sugars as signaling molecules is well illustrated by the variety of sugar

Expression and evolutionary features of the hexokinase gene family in Arabidopsis.

Arabidopsis hexokinase1 (HXK1) is a moonlighting protein that has separable functions in glucose signaling and in glucose metabolism. In this study, we have characterized expression features and glucose phosphorylation activities of the six HXK gene family members in Arabidopsis thaliana. Three of the genes encode catalytically active proteins, including a stromal-localized HXK3 protein that is expressed mostly in sink organs. We also show that three of the genes encode hexokinase-like (HKL) proteins, which are about 50% identical to AtHXK1, but do not phosphorylate glucose or fructose. Expression studies indicate that both HKL1 and HKL2 transcripts occur in most, if not all, plant tissues and that both proteins are targeted within cells to mitochondria. The HKL1 and HKL2 proteins have 6–10 amino acid insertions/deletions (indels) at the adenosine binding domain. In contrast, HKL3 transcript was detected only in flowers, the protein lacks the noted indels, and the protein has many other amino acid changes that might compromise its ability even to bind glucose or ATP. Activity measurements of HXKs modified by site-directed mutagenesis suggest that the lack of catalytic activities in the HKL proteins might be attributed to any of numerous existing changes. Sliding windows analyses of coding sequences in A. thaliana and A. lyrata ssp. lyrata revealed a differential accumulation of nonsynonymous changes within exon 8 of both HKL1 and HXK3 orthologs. We further discuss the possibility that the non-catalytic HKL proteins have regulatory functions instead of catalytic functions.

Evolutionary Lineages and Functional Diversification of Plant Hexokinases

Sequencing data from 10 species show that a plant hexokinase (HXK) family contains 5-11 genes. Functionally, a given family can include metabolic catalysts, glucose signaling proteins, and non-catalytic, apparent regulatory enzyme homologs. This study has two goals. The first aim is to develop a predictive method to determine which HXK proteins within a species have which type of function. The second aim is to determine whether HXK-dependent glucose signaling proteins occur among more primitive plants, as well as among angiosperms. Using a molecular phylogeny approach, combined with selective experimental testing, we found that non-catalytic HXK homologs might occur in all plants, including the relatively primitive Selaginella moellendorffi. We also found that different lineages of angiosperm HXKs have apparent conserved features for catalytic activity and for sub-cellular targeting. Most higher-plant HXKs are expressed predominantly at mitochondria, with HXKs of one lineage occurring in the plastid, and HXKs of one monocot lineage occurring in the cytosol. Using protoplast transient expression assays, we found that HXK glucose signaling proteins occur likely in all higher plants and in S. moellendorffi as well. Thus, the use of glucose by plant HXK isoforms in metabolism and/or as a regulatory metabolite occurs as widespread, conserved processes.

Plant sugar sensing and signaling – a complex reality

In plants, sugars function as a metabolic resource, but they are also important regulators of many processes associated with growth, maturation and senescence1xCarbohydrate-modulated gene expression in plants. Koch, K.E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 509–540CrossRef | PubMedSee all References, 2xSugar sensing in higher plants. Jang, J-C. and Sheen, J.S. Trends Plant Sci. 1997; 2: 208–214Abstract | Full Text PDFSee all References. Their regulatory activities include both the repression and activation of many genes, and it is probable that several distinct sensing and transduction mechanisms are involved2xSugar sensing in higher plants. Jang, J-C. and Sheen, J.S. Trends Plant Sci. 1997; 2: 208–214Abstract | Full Text PDFSee all References, 3xSugar sensing and sugar-mediated signal transduction in plants. Smeekens, S. and Rooks, F. Plant Physiol. 1997; 115: 7–13PubMedSee all References, 4xSucrose is a signal molecule in assimilate partitioning. Chiou, T-J. and Bush, D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4784–4788CrossRef | PubMed | Scopus (258)See all References. In response to a recent article by Nigel Halford and colleagues5xIs hexokinase really a sugar sensor in plants?. Halford, N.G., Purcell, P.C., and Hardie, D.G. Trends Plant Sci. 1999; 4: 117–120Abstract | Full Text | Full Text PDF | PubMed | Scopus (104)See all References5, which questioned whether hexokinase functions in sugar sensing and signaling, as well as in hexose metabolism, we consider evidence that substantiates this dual function. We also highlight some of the differences in sugar signaling between yeast and plants, which emphasize the unique ways in which plants have evolved to carry out their life cycle.Using both genetic and biochemical approaches, there is much available evidence that plant hexokinase-dependent glucose metabolism can be separated from hexokinase-dependent glucose signaling.Firstly, transformation of Arabidopsis for sense or antisense expression of AtHXK1 or AtHXK2 resulted in hyper- and hypo-sensitive plants, respectively, based on rbcs and cab gene expression and on seedling bioassays with exogenous sugars6xHexokinase as a sugar sensor in higher plants. Jang, J-C. et al. Plant Cell. 1997; 9: 5–19PubMedSee all References6. Importantly, this effect was not caused by altered HXK-dependent glucose metabolism, because Arabidopsis transformed with yeast HXK2 were actually less sensitive to glucose repression, in spite of a three- to fivefold increase in enzyme activity.Secondly, glucose analogs can arrest seed germination and/or seedling development by a mechanism that affects sugar signaling independently of hexose metabolism. For example, low levels of mannose can block germination of Arabidopsis without affecting seed levels of ATP or inorganic phosphate7xMannose inhibits Arabidopsis germination via a hexokinase-mediated step. Pego, J.V., Weisbeek, P.J., and Smeekens, S.C.M. Plant Physiol. 1999; 119: 1017–1023CrossRef | PubMedSee all References7, and mannose-dependent repression of germination can be overcome by mannoheptulose (a competitive inhibitor of hexokinase)7xMannose inhibits Arabidopsis germination via a hexokinase-mediated step. Pego, J.V., Weisbeek, P.J., and Smeekens, S.C.M. Plant Physiol. 1999; 119: 1017–1023CrossRef | PubMedSee all References7. Furthermore, germination in the presence of mannoheptulose indicates that hexose phosphorylation by hexokinase is not required for germination of Arabidopsis.Finally, glucose repression of gene expression has also been examined using freshly isolated mesophyll protoplasts. Co-transfection of hexokinase substrates, glucose analogs or intermediary metabolites with reporter genes, has demonstrated: gene specificity; a requirement for hexose phosphorylation; and the lack of inhibition by hexose-phosphates, other glycolytic intermediates, ATP or inorganic phosphate8xSugar sensing in higher plants. Jang, J.C. and Sheen, J.S. Plant Cell. 1994; 6: 1665–1679PubMedSee all References8.These and other available data (e.g. 3xSugar sensing and sugar-mediated signal transduction in plants. Smeekens, S. and Rooks, F. Plant Physiol. 1997; 115: 7–13PubMedSee all References, 9xCarbon catabolite repression regulates glyoxylate cycle gene expression in cucumber. Graham, I.A., Denby, K.J., and Leaver, C.J. Plant Cell. 1994; 6: 761–772PubMedSee all References) offer compelling and unequivocal evidence that plant hexokinase plays a major role in glucose-dependent modulation of gene expression and plant growth. The recent isolation of two AtHXK1 mutants has provided definitive evidence for such a role (L. Zhou et al., unpublished), but a complete understanding of this role awaits a detailed molecular determination of the encoded proteins putative signaling and metabolic functions. The isolation of suppressors in a plant hexokinase-null mutant, or the isolation of HXK mutants that can uncouple the two functions, will prove that the two roles are indeed separable.Recent research has indicated that sugar signaling in plants also occurs by a hexose-dependent but hexokinase-independent pathway10xGlucose and stress independently regulate source and sink metabolism and defense mechanisms via signal transduction pathways involving protein phosphorylation. Ehness, R. et al. Plant Cell. 1997; 9: 1825–1841PubMedSee all References10, as well as by a sucrose-dependent pathway4xSucrose is a signal molecule in assimilate partitioning. Chiou, T-J. and Bush, D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4784–4788CrossRef | PubMed | Scopus (258)See all References4. The existence of multiple pathways indicates that sugar-signal transduction processes are relatively complex in plants. It has recently been suggested that plant homologs of yeast SNF1 kinase mediate both hexose and sucrose signaling mechanisms, largely at the exclusion of any role for hexokinase5xIs hexokinase really a sugar sensor in plants?. Halford, N.G., Purcell, P.C., and Hardie, D.G. Trends Plant Sci. 1999; 4: 117–120Abstract | Full Text | Full Text PDF | PubMed | Scopus (104)See all References5. However, if these homologs are of central importance, then it remains to be explained why antisense expression of the gene in potato only appears to affect the expression of sucrose synthase5xIs hexokinase really a sugar sensor in plants?. Halford, N.G., Purcell, P.C., and Hardie, D.G. Trends Plant Sci. 1999; 4: 117–120Abstract | Full Text | Full Text PDF | PubMed | Scopus (104)See all References5.We believe that the unique properties of plant carbohydrate biochemistry, and its interface with multiple growth processes, will necessarily lead to different models for sugar signaling in plants compared with yeast. First, in yeast, glucose-dependent repression of gene expression involves prominent negative DNA regulatory elements that control the utilization of non-fermentable carbohydrates11xGlucose repression in fungi. Ronne, H. Trends Genet. 1995; 11: 12–17Abstract | Full Text PDF | PubMed | Scopus (321)See all References11. In contrast, plant sugar repression involves positive DNA regulatory elements, which in source tissues respond to inputs from carbohydrate biosynthesis (‘end-product inhibition’) and in sink tissues respond to carbohydrate utilization12xMetabolic repression of transcription in higher plants. Sheen, J.S. Plant Cell. 1990; 2: 1027–1038PubMedSee all References12. Second, the trafficking of sugars is very different in plant cells and yeast and, as such, the relationships between sugar signaling and sugar metabolism will also be different. Plant carbohydrate metabolism involves intracellular cycles, extensive compartmentation, and partitioning to, or from, organ-level transport processes13xSee all References13. All these processes are modulated by very sophisticated, diurnal regulatory programs. Finally, plant sugar signaling pathways can involve crosstalk with hormone signaling pathways that control growth and development and are unique to plants14xGlucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant. Zhou, L. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10294–10299CrossRef | PubMed | Scopus (282)See all References14. This type of complex signaling network in a multicellular plant does not occur in the unicellular yeast. Thus, it might be inappropriate to largely restrict plant sugar signaling models to known paradigms for yeast sugar signaling5xIs hexokinase really a sugar sensor in plants?. Halford, N.G., Purcell, P.C., and Hardie, D.G. Trends Plant Sci. 1999; 4: 117–120Abstract | Full Text | Full Text PDF | PubMed | Scopus (104)See all References5. Rather, it might be better to recognize that evolutionary motifs often involve a ‘used once, borrowed twice’ scenario.

Arabidopsis Hexokinase-Like1 and Hexokinase1 Form a Critical Node in Mediating Plant Glucose and Ethylene Responses

Arabidopsis (Arabidopsis thaliana) Hexokinase-Like1 (HKL1) lacks glucose (Glc) phosphorylation activity and has been shown to act as a negative regulator of plant growth. Interestingly, the protein has a largely conserved Glc-binding domain, and protein overexpression was shown previously to promote seedling tolerance to exogenous 6% (w/v) Glc. Since these phenotypes occur independently of cellular Glc signaling activities, we have tested whether HKL1 might promote cross talk between the normal antagonists Glc and ethylene. We show that repression by 1-aminocyclopropane-1-carboxylic acid (ACC) of the Glc-dependent developmental arrest of wild-type Arabidopsis seedlings requires the HKL1 protein. We also describe an unusual root hair phenotype associated with growth on high Glc medium that occurs prominently in HKL1 overexpression lines and in glucose insensitive 2-1 (gin2-1), a null mutant of Hexokinase1 (HXK1). Seedlings of these lines produce bulbous root hairs with an enlarged base after transfer from agar plates with normal medium to plates with 6% Glc. Seedling transfer to plates with 2% Glc plus ACC mimics the high-Glc effect in the HKL1 overexpression line but not in gin2-1. A similar ACC-stimulated, bulbous root hair phenotype also was observed in wild-type seedlings transferred to plates with 9% Glc. From transcript expression analyses, we found that HKL1 and HXK1 have differential roles in Glc-dependent repression of some ethylene biosynthesis genes. Since we show by coimmunoprecipitation assays that HKL1 and HXK1 can interact, these two proteins likely form a critical node in Glc signaling that mediates overlapping, but also distinct, cellular responses to Glc and ethylene treatments.

Function of Arabidopsis hexokinase-like1 as a negative regulator of plant growth

A recent analysis of the hexokinase (HXK) gene family from Arabidopsis revealed that three hexokinase-like (HKL) proteins lack catalytic activity, but share about 50% identity with the primary glucose (glc) sensor/transducer protein AtHXK1. Since the AtHKL1 protein is predicted to bind glc, although with a relatively decreased affinity, a reverse genetics approach was used to test whether HKL1 might have a related regulatory function in plant growth. By comparing phenotypes of an HKL1 mutant (hkl1-1), an HXK1 mutant (gin2-1), and transgenic lines that overexpress HKL1 in either wild-type or gin2-1 genetic backgrounds, it is shown that HKL1 is a negative effector of plant growth. Interestingly, phenotypes of HKL1 overexpression lines are generally very similar to those of gin2-1. These are quantified, in part, as reduced seedling sensitivity to high glc concentrations and reduced seedling sensitivity to auxin-induced lateral root formation. However, commonly recognized targets of glc signalling are not apparently altered in any of the HKL1 mutant or transgenic lines. In fact, most, but not all, of the observed phenotypes associated with HKL1 overexpression occur independently of the presence of HXK1 protein. The data indicate that HKL1 mediates cross-talk between glc and other plant hormone response pathways. It is also considered Whether a possibly decreased glc binding affinity of HKL1 could possibly be a feedback mechanism to limit plant growth in the presence of excessive carbohydrate availability is further considered.

Characterization of Rubisco activase from thermally contrasting genotypes of Acer rubrum (Aceraceae).

The lability of Rubisco activase function is thought to have a major role in the decline of leaf photosynthesis under moderate heat (<35°C). To investigate this further, we characterized Rubisco activase and explored its role in the previously demonstrated thermal acclimation and inhibition of two genotypes of Acer rubrum originally collected from Florida (FL) and Minnesota (MN). When plants were grown at 33/25°C (day/night) for 21 d, the FL genotype compared to the MN genotype maintained about a two-fold increase in leaf photosynthetic rates at 33-42°C and had a 22% increase in the maximal rate of Rubisco carboxylation (V(cmax)) at 33°C under nonphotorespiratory conditions. Both genotypes had two leaf Rca transcripts, likely from equivalent alternative splicing events. The RCA1 and RCA2 proteins increased modestly in FL plants under warmer temperature, while only RCA2 protein increased in MN plants. Rubisco large subunit (RbsL) protein abundance was relatively unaffected in either genotype by temperature. These results support the idea that Rubisco activase, particularly the ratio of Rubisco activase to Rubisco, may play a role in the photosynthetic heat acclimation in A. rubrum and may have adaptive significance. This mechanism alone is not likely to entirely explain the thermotolerance in the FL genotype, and future research on adaptive mechanisms to high temperatures should consider activase function in a multipathway framework.

Actin-based cellular framework for glucose signaling by Arabidopsis hexokinase1.

Glucose functions in plants both as a metabolic resource as well as a hormone that regulates expression of many genes. Arabidopsis hexokinase1 (HXK1) is the best understood plant glucose sensor/transducer, yet we are only now appreciating the cellular complexity of its signaling functions. We have recently shown that one of the earliest detectable responses to plant glucose treatments are extensive alterations of cellular F-actin. Interestingly, AtHXK1 is predominantly located on mitochondria, yet also can interact with actin. A normal functioning actin cytoskeleton is required for HXK1 to act as an effector in glucose signaling assays. We have suggested that HXK1 might alter F-actin dynamics and thereby influence the formation and/or stabilization of cytoskeleton-bound polysomes. In this Addendum, we have extended our initial observations on the subcellular targeting of HXK1 and its interaction with F-actin. We then further consider the cellular context in which HXK1 might regulate gene expression.

Symbol addition by monkeys provides evidence for normalized quantity coding

Significance Symbol-literate monkeys can be trained to combine, or add, pairs of large numbers. They transfer to a novel symbol set, ruling out memorization of each symbol pair. Their addition behavior indicates an underlying relative scaling of magnitude. Weber’s law can be explained either by a compressive scaling of sensory response with stimulus magnitude or by a proportional scaling of response variability. These two mechanisms can be distinguished by asking how quantities are added or subtracted. We trained Rhesus monkeys to associate 26 distinct symbols with 0–25 drops of reward, and then tested how they combine, or add, symbolically represented reward magnitude. We found that they could combine symbolically represented magnitudes, and they transferred this ability to a novel symbol set, indicating that they were performing a calculation, not just memorizing the value of each combination. The way they combined pairs of symbols indicated neither a linear nor a compressed scale, but rather a dynamically shifting, relative scaling.