Volume 3, Issue 2, Pages 334-346 (March 2010) Evolutionary Lineages and Functional Diversification of Plant Hexokinases  Karve Rucha , Lauria Michael , Virnig Annie , Xia Xiaoxia , Rauh Bradley L. , Moore Brandon d.   Molecular Plant  Volume 3, Issue 2, Pages 334-346 (March 2010) DOI: 10.1093/mp/ssq003 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Molecular Phylogeny of Plant HXKs. The phylogenetic tree was generated from aligned sequences of predicted proteins from 70 plant HXK genes. The outgroup consisted of HXK1 from V. carteri plus HXK1 and HXK2 from S. cerevisiae. The tree was made by maximum likelihood analysis using 2000 bootstrap predictions and 50% majority rule (PhyML; see Methods for further details). Group designations were made from visual inspection of apparent clusters. Group 3 includes HKL proteins thought to bind glc, while Group 5 includes ones thought not to bind glc. The tree topology indicates there are independent primitive plant and higher plant lineages related by common descent from an unidentified ancestral protein. Similar topologies were obtained using sequences containing only the major domains (glc binding domain + L4 loop; adenosine binding domain) or sequences lacking the more variable N-termini (data not shown). Pp, Physcomitrella patens; At, Arabidopsis thaliana; Os, Oryza sativa; Pt, Populus trichocarpa; Rc, Ricinus communis; Sb, Sorghum bicolor; Sc, Saccharomyces cerevisiae; Sm, Selaginella moellendorffii; Vc, Volvox carteri; Vv, Vitis vinifera; Zm, Zea mays. Molecular Plant 2010 3, 334-346DOI: (10.1093/mp/ssq003) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 The Phylogenetic Group 3 HKL Proteins Have a Unique Indel. (A) Partial sequences flanking the indel of HXKs from two eudicot species and two monocot species from phylogenetic Group 3 were aligned with the homologous region of HXKs from the same species present in Groups 6 and 7. In AtHXK1, the partial sequence shown above begins at R-405. The indel is located proximal to the N-terminal side of the adenosine domain (the latter starting at T-425 in AtHXK1; Karve et al., 2008). At, A. thaliana; Pt, P. trichocarpa; Os, O. sativa; Sb, S. bicolor. (B) The indel identified in AtHKL1 forms an extended low-complexity region. Shown are diagrams of predicted 2° structures for AtHXK1 and AtHKL1 (PSIPRED; McGuffin et al., 2000), which begin at amino acid 401 in each case and which show the regions flanking the indel of AtHKL1 (G-431 through R-439) or the homologous position in AtHXK1. The green cylinders show parts of α-helices and the yellow arrows show β-sheets. (C) Lengths of the homologous low-complexity region from the different phylogenetic groups of angiosperms (see Figure 1). Values are the mean number of amino acids within the low-complexity region, ± 1 SD. For Groups 4, 6, and 7, n = 37; for Group 3, n = 13; and for Group 5, n = 4. Molecular Plant 2010 3, 334-346DOI: (10.1093/mp/ssq003) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Non-Catalytic HXK Family Proteins Occur in Diverse Taxa, Including S. moellendorffii. (A) Glucokinase activities were measured after transient expression of corresponding cDNAs in maize protoplasts. Assays were done generally for 30 min. Values are normalized between experiments according to the activity of transfected AtHXK1. Values are means ± 1 SD (n = 3–6). The activity measured from non-transfected control protoplasts represents endogenous, glc phosphorylation activity. Since the SD value for control protoplasts was 0.5%, our detection limit is about 1% of normalized AtHXK1 activity. (B) Immunoblot of expressed HXK family proteins in maize protoplasts. Membranes were probed with anti-Flag M2 primary antibody. Predicted molecular weights range from about 51 kD (SmHXK5) to 56 kD (SmHXK2). Molecular Plant 2010 3, 334-346DOI: (10.1093/mp/ssq003) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Selected HXK–GFP Fusion Proteins Are Expressed in the Cytosol or at Mitochondria. Plasmid cDNAs were transfected into pea protoplasts and their expression imaged by fluorescence microscopy after 6–8 h. Not shown are equivalent results obtained using maize protoplasts. SmHXK3 and SmHXK5 are from phylogenetic Group 2 (each expressed in the cytosol); SbHXK3 is from Group 3 (mitochondria); SbHXK8 is from Group 7A (cytosol); PtHXK1 is from Group 6 (mitochondria); and ScHXK2 is shown as a positive control for a cytosol-localized HXK (Balasubramanian et al., 2007). Sm, Selaginella moellendorffii; Sb, Sorghum bicolor; Pt, Populus trichocarpa; Sc, Saccharomyces cerevisiae. Molecular Plant 2010 3, 334-346DOI: (10.1093/mp/ssq003) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Glc Signaling and Non-Signaling HXKs Occur in Diverse Plant Taxa. Protoplasts were co-transfected with pRBCS–LUC and an internal control, pUBQ10–GUS, plus different HXKs as effector proteins. After 90 min from transfection, protoplasts were treated with 0.25 mM glc. Values are means of three to six biological replicates, ± SD, of the relative LUC units to GUS activities, after normalizing to the control treatment (no glc, no effector). GUS activity was not affected by glc treatment. Relative LUC/GUS values with AtHKL1 and SbHXK3 were not significantly different from the control value. See Figure 3B for immunoblot of expressed protein. Molecular Plant 2010 3, 334-346DOI: (10.1093/mp/ssq003) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions