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.

Highly selective directed arylation reactions via back-to-back dehydrogenative C-H borylation/arylation reactions.

Dimeric rhodium N-heterocyclic carbene complexes are demonstrated to be effective catalyst precursors for directed C-H borylation reactions at room temperature. The reactions are highly selective for mono-borylation and can be combined with a one-pot Suzuki-Miyaura coupling to give C-H arylation products with exclusive selectivity for mono-arylation without the requirement for steric blocking groups.

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.

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.

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.

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.

A role for F-actin in hexokinase-mediated glucose signaling.

HEXOKINASE1 (HXK1) from Arabidopsis (Arabidopsis thaliana) has dual roles in glucose (Glc) signaling and in Glc phosphorylation. The cellular context, though, for HXK1 function in either process is not well understood. Here we have shown that within normal experimental detection limits, AtHXK1 is localized continuously to mitochondria. Two mitochondrial porin proteins were identified as capable of binding to overexpressed HXK1 protein, both in vivo and in vitro. We also found that AtHXK1 can be associated with its structural homolog, F-actin, based on their coimmunoprecipitation from transgenic plants that overexpress HXK1-FLAG or from transient expression assays, and based on their localization in leaf cells after cryofixation. This association might be functionally important because Glc signaling in protoplast transient expression assays is compromised by disruption of F-actin. We also demonstrate that Glc treatment of Arabidopsis seedlings rapidly and reversibly disrupts fine mesh actin filaments. The possible roles of actin in HXK-dependent Glc signaling are discussed.