Dependence on clade II bHLH transcription factors for nursing of haploid products by tapetal-like cells is conserved between moss sporangia and angiosperm anthers.

Clade II basic helix-loop-helix transcription factors (bHLH TFs) are essential for pollen production and tapetal nursing functions in angiosperm anthers. As pollen has been suggested to be related to bryophyte spores by descent, we characterized two Physcomitrium (Physcomitrella) patens clade II bHLH TFs (PpbHLH092 and PpbHLH098), to test if regulation of sporogenous cells and the nursing cells surrounding them is conserved between angiosperm anthers and bryophyte sporangia. We made CRISPR-Cas9 reporter and loss-of-function lines to address the function of PpbHLH092/098. We sectioned and analyzed WT and mutant sporophytes for a comprehensive stage-by-stage comparison of sporangium development. Spore precursors in the P. patens sporangium are surrounded by nursing cells showing striking similarities to tapetal cells in angiosperms. Moss clade II bHLH TFs are essential for the differentiation of these tapetal-like cells and for the production of functional spores. Clade II bHLH TFs provide a conserved role in controlling the sporophytic somatic cells surrounding and nursing the sporogenous cells in both moss sporangia and angiosperm anthers. This supports the hypothesis that such nursing functions in mosses and angiosperms, lineages separated by c. 450 million years, are related by descent.

MS1/MMD1 homologues in the moss Physcomitrium patens are required for male and female gametogenesis.

The Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. This pollen-based male gametogenesis is a derived trait of seed plants. Male gametogenesis in the common ancestors of land plants is instead likely to have been reminiscent of that in extant bryophytes where flagellated sperms are produced by an elaborate gametophyte generation. Still, also bryophytes possess MS1/MMD1-related PHD proteins. We addressed the function of two MS1/MMD1-homologues in the bryophyte model moss Physcomitrium patens by the generation and analysis of reporter and loss-of-function lines. The two genes are together essential for both male and female fertility by providing functions in the gamete-producing inner cells of antheridia and archegonia. They are furthermore expressed in the diploid sporophyte generation suggesting a function during sporogenesis, a process proposed related by descent to pollen formation in angiosperms. We propose that the moss MS1/MMD1-related regulatory network required for completion of male and female gametogenesis, and possibly for sporogenesis, represent a heritage from ancestral land plants.

The Physcomitrium patens egg cell expresses several distinct epigenetic components and utilizes homologues of BONOBO genes for cell specification.

Although land plant germ cells have received much attention, knowledge about their specification is still limited. We thus identified transcripts enriched in egg cells of the bryophyte model species Physcomitrium patens, compared the results with angiosperm egg cells, and selected important candidate genes for functional analysis. We used laser-assisted microdissection to perform a cell-type-specific transcriptome analysis on egg cells for comparison with available expression profiles of vegetative tissues and male reproductive organs. We made reporter lines and knockout mutants of the two BONOBO (PbBNB) genes and studied their role in reproduction. We observed an overlap in gene activity between bryophyte and angiosperm egg cells, but also clear differences. Strikingly, several processes that are male-germline specific in Arabidopsis are active in the P. patens egg cell. Among those were the moss PbBNB genes, which control proliferation and identity of both female and male germlines. Pathways shared between male and female germlines were most likely present in the common ancestors of land plants, besides sex-specifying factors. A set of genes may also be involved in the switches between the diploid and haploid moss generations. Nonangiosperm gene networks also contribute to the specification of the P. patens egg cell.

Selective auxin agonists induce specific AUX/IAA protein degradation to modulate plant development.

Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCFTIR1/AFB functionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses.

Studies of moss reproductive development indicate that auxin biosynthesis in apical stem cells may constitute an ancestral function for focal growth control.

The plant hormone auxin is a key factor for regulation of plant development, and this function was probably reinforced during the evolution of early land plants. We have extended the available toolbox to allow detailed studies of how auxin biosynthesis and responses are regulated in moss reproductive organs, their stem cells and gametes to better elucidate the function of auxin in the morphogenesis of early land plants. We measured auxin metabolites and identified IPyA (indole-3-pyruvic acid) as the main biosynthesis pathway in Physcomitrium (Physcomitrella) patens and established knock-out, overexpressor and reporter lines for biosynthesis genes which were analyzed alongside previously reported auxin-sensing and transport reporters. Vegetative and reproductive apical stem cells synthesize auxin. Sustained stem cell activity depends on an inability to sense the auxin produced while progeny of the stem cells respond to the auxin, aiding in the control of cell division, expansion and differentiation. Gamete precursors are dependent on a certain degree of auxin sensing, while the final differentiation is a low auxin-sensing process. Tha data presented indicate that low auxin activity may represent a conserved hallmark of land plant gametes, and that local auxin biosynthesis in apical stem cells may be part of an ancestral mechanism to control focal growth.

Minimal auxin sensing levels in vegetative moss stem cells revealed by a ratiometric reporter.

Efforts to reveal ancestral functions of auxin, a key regulator of plant growth and development, and its importance for evolution have been hampered by a fragmented picture of auxin response domains in early-diverging land plants. We report the mapping of auxin sensing and responses during vegetative moss development using novel reporters. We established a moss-specific ratiometric reporter (PpR2D2) for Auxin Response Element- and AUXIN RESPONSE FACTOR-independent auxin sensing in Physcomitrella patens, and its readout during vegetative development was compared with new promoter-based GmGH3::GFPGUS and DR5revV2::GFPGUS auxin response reporters. The ratiometric reporter responds rapidly to auxin in a time-, dose- and TRANSPORT INHIBITOR RESISTANT1/AUXIN F-BOX-dependent manner and marks known, anticipated and novel auxin sensing domains. It reveals proximal auxin sensing maxima in filamentous tissues and sensing minima in all five vegetative gametophytic stem cell types as well as dividing cells. PpR2D2 readout is compliant with an ancestral function of auxin as a positive regulator of differentiation vs proliferation in stem cell regions. The PpR2D2 reporter is a sensitive tool for high-resolution mapping of auxin sensing, which can increase our knowledge of auxin function in early-diverging land plants substantially, thereby advancing our understanding of its importance for plant evolution.

Auxin-mediated developmental control in the moss Physcomitrella patens.

The signalling molecule auxin regulates many fundamental aspects of growth and development in plants. We review and discuss what is known about auxin-regulated development in mosses, with special emphasis on the model species Physcomitrella patens. It is well established that mosses and other early diverging plants produce and respond to auxin. By sequencing the P. patens genome, it became clear that it encodes many core proteins important for auxin homeostasis, perception, and signalling, which have also been identified in flowering plants. This suggests that the auxin molecular network was present in the last common ancestor of flowering plants and mosses. Despite fundamental differences in their life cycles, key processes such as organ initiation and outgrowth, branching, tropic responses, as well as cell differentiation, division, and expansion appear to be regulated by auxin in the two lineages. This knowledge paves the way for studies aimed at a better understanding of the origin and evolution of auxin function and how auxin may have contributed to the evolution of land plants.

The MOSS Physcomitrella patens reproductive organ development is highly organized, affected by the two SHI/STY genes and by the level of active auxin in the SHI/STY expression domain.

In order to establish a reference for analysis of the function of auxin and the auxin biosynthesis regulators SHORT INTERNODE/STYLISH (SHI/STY) during Physcomitrella patens reproductive development, we have described male (antheridial) and female(archegonial) development in detail, including temporal and positional information of organ initiation. This has allowed us to define discrete stages of organ morphogenesis and to show that reproductive organ development in P. patens is highly organized and that organ phyllotaxis differs between vegetative and reproductive development. Using the PpSHI1 and PpSHI2 reporter and knockout lines, the auxin reporters GmGH3(pro):GUS and PpPINA(pro):GFP-GUS, and the auxin-conjugating transgene PpSHI2(pro):IAAL, we could show that the PpSHI genes, and by inference also auxin, play important roles for reproductive organ development in moss. The PpSHI genes are required for the apical opening of the reproductive organs, the final differentiation of the egg cell, and the progression of canal cells into a cell death program. The apical cells of the archegonium, the canal cells, and the egg cell are also sites of auxin responsiveness and are affected by reduced levels of active auxin, suggesting that auxin mediates PpSHI function in the reproductive organs.

Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

The Arabidopsis thaliana STYLISH1 protein acts as a transcriptional activator regulating auxin biosynthesis.

The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

PIF-independent regulation of growth by an evening complex in the liverwort Marchantia polymorpha.

Previous studies in the liverwort Marchantia polymorpha have shown that the putative evening complex (EC) genes LUX ARRHYTHMO (LUX) and ELF4-LIKE (EFL) have a function in the liverwort circadian clock. Here, we studied the growth phenotypes of MpLUX and MpEFL loss-of-function mutants, to establish if PHYTOCHROME-INTERACTING FACTOR (PIF) and auxin act downstream of the M. polymorpha EC in a growth-related pathway similar to the one described for the flowering plant Arabidopsis. We examined growth rates and cell properties of loss-of-function mutants, analyzed protein-protein interactions and performed gene expression studies using reporter genes. Obtained data indicate that an EC can form in M. polymorpha and that this EC regulates growth of the thallus. Altered auxin levels in Mplux mutants could explain some of the phenotypes related to an increased thallus surface area. However, because MpPIF is not regulated by the EC, and because Mppif mutants do not show reduced growth, the growth phenotype of EC-mutants is likely not mediated via MpPIF. In Arabidopsis, the circadian clock regulates elongation growth via PIF and auxin, but this is likely not an evolutionarily conserved growth mechanism in land plants. Previous inventories of orthologs to Arabidopsis clock genes in various plant lineages showed that there is high levels of structural differences between clocks of different plant lineages. Here, we conclude that there is also variation in the output pathways used by the different plant clocks to control growth and development.

Apical dominance control by TAR-YUC-mediated auxin biosynthesis is a deep homology of land plants.

A key aim in biology is to identify which genetic changes contributed to the evolution of form through time. Apical dominance, the inhibitory effect exerted by shoot apices on the initiation or outgrowth of distant lateral buds, is a major regulatory mechanism of plant form.1 Nearly a century of studies in the sporophyte of flowering plants have established the phytohormone auxin as a front-runner in the search for key factors controlling apical dominance,2,3 identifying critical roles for long-range polar auxin transport and local auxin biosynthesis in modulating shoot branching.4-10 A capacity for lateral branching evolved by convergence in the gametophytic shoot of mosses and primed its diversification;11 however, polar auxin transport is relatively unimportant in this developmental process,12 the contribution of auxin biosynthesis genes has not been assessed, and more generally, the extent of conservation in apical dominance regulation within the land plants remains largely unknown. To fill this knowledge gap, we sought to identify genetic determinants of apical dominance in the moss Physcomitrium patens. Here, we show that leafy shoot apex decapitation releases apical dominance through massive and rapid transcriptional reprogramming of auxin-responsive genes and altering auxin biosynthesis gene activity. We pinpoint a subset of P. patens TRYPTOPHAN AMINO-TRANSFERASE (TAR) and YUCCA FLAVIN MONOOXYGENASE-LIKE (YUC) auxin biosynthesis genes expressed in the main and lateral shoot apices and show that they are essential for coordinating branch initiation and outgrowth. Our results demonstrate that local auxin biosynthesis acts as a pivotal regulator of apical dominance in moss and constitutes a shared mechanism underpinning shoot architecture control in land plants.

Two novel types of hexokinases in the moss Physcomitrella patens.

BACKGROUND: Hexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively. RESULTS: We have now characterized all 11 hexokinase encoding genes in Physcomitrella. Based on their N-terminal sequences and intracellular localizations, three of the encoded proteins are type A hexokinases and four are type B hexokinases. One of the type B hexokinases has a splice variant without a membrane anchor, that localizes to the cytosol and the nucleus. However, we also found two new types of hexokinases with no obvious orthologs in vascular plants. Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus. Type D hexokinases, encoded by three genes, have membrane anchors and localize to mitochondrial membranes, but their sequences differ from those of the type B hexokinases. Interestingly, all moss hexokinases are more similar to each other in overall sequence than to hexokinases from other plants, even though characteristic sequence motifs such as the membrane anchor of the type B hexokinases are highly conserved between moss and vascular plants, indicating a common origin for hexokinases of the same type. CONCLUSIONS: We conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants. In particular, the presence of a cytosolic and nuclear hexokinase (type C) sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinase gene family in Physcomitrella has undergone concerted evolution.

Quickly-released peroxidase of moss in defense against fungal invaders.

Mosses (Bryophyta) are nonvascular plants that constitute a large part of the photosynthesizing biomass and carbon storage on Earth. Little is known about how this important portion of flora maintains its health status. This study assessed whether the moss, Physcomitrella patens, responds to treatment with chitosan, a fungal cell wall-derived compound inducing defense against fungal pathogens in vascular plants. Application of chitosan to liquid culture of P. patens caused a rapid increase in peroxidase activity in the medium. For identification of the peroxidase(s), matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/MS, other methods and the whole-genome sequence of P. patens were utilized. Peroxidase gene knock-out mutants were made and inoculated with fungi. The peroxidase activity resulted from a single secreted class III peroxidase (Prx34) which belonged to a P. patens specific phylogenetic cluster in analysis of the 45 putative class III peroxidases of P. patens and those of Arabidopsis and rice. Saprophytic and pathogenic fungi isolated from another moss killed the Prx34 knockout mutants but did not damage wild-type P. patens. The data point out the first specific host factor that is pivotal for pathogen defense in a nonvascular plant. Furthermore, results provide conclusive evidence that class III peroxidases in plants are needed in defense against hostile invasion by fungi.

Autophagy is required for gamete differentiation in the moss Physcomitrella patens.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Directional auxin transport mechanisms in early diverging land plants.

The emergence and radiation of multicellular land plants was driven by crucial innovations to their body plans. The directional transport of the phytohormone auxin represents a key, plant-specific mechanism for polarization and patterning in complex seed plants. Here, we show that already in the early diverging land plant lineage, as exemplified by the moss Physcomitrella patens, auxin transport by PIN transporters is operational and diversified into ER-localized and plasma membrane-localized PIN proteins. Gain-of-function and loss-of-function analyses revealed that PIN-dependent intercellular auxin transport in Physcomitrella mediates crucial developmental transitions in tip-growing filaments and waves of polarization and differentiation in leaf-like structures. Plasma membrane PIN proteins localize in a polar manner to the tips of moss filaments, revealing an unexpected relation between polarization mechanisms in moss tip-growing cells and multicellular tissues of seed plants. Our results trace the origins of polarization and auxin-mediated patterning mechanisms and highlight the crucial role of polarized auxin transport during the evolution of multicellular land plants.

Sesquiterpenes from the conifer root rot pathogen Heterobasidion occidentale.

Investigation of the production of secondary metabolites of Heterobasidion occidentale led to the isolation and identification of six sesquiterpenes (illudolone A and B, illudolactone A and B, deoxyfomannosin A and B) along with the well-known sesquiterpene fomannosin and the previously described benzohydrofuran fomannoxin. The structures and relative configurations of the compounds were determined by 1D and 2D NMR spectroscopic analysis as well as by HRMS. Their absolute configuration and biosynthesis were suggested and discussed in relation to fomannosin. Four compounds showed growth inhibiting activity against several basidiomycetes, Phlebiopsis gigantea, Phanerochaete chrysosporium and H. occidentale, and toxicity towards the moss Physcomitrella patens. In addition, one compound displayed activity against the bacterium Variovorax paradoxus as well as against the ascomycete Fusarium oxysporum.

The moss genes PpSKI1 and PpSKI2 encode nuclear SnRK1 interacting proteins with homologues in vascular plants.

The yeast Snf1, animal AMPK, and plant SnRK1 protein kinases constitute a family of related proteins that have been proposed to serve as metabolic sensors of the eukaryotic cell. We have previously reported the characterization of two redundant SnRK1 encoding genes (PpSNF1a and PpSNF1b) in the moss Physcomitrella patens. Phenotypic analysis of the snf1a snf1b double knockout mutant suggested that SnRK1 is important for the plant's ability to recognize and adapt to conditions of limited energy supply, and also suggested a possible role of SnRK1 in the control of plant development. We have now used a yeast two-hybrid system to screen for PpSnf1a interacting proteins. Two new moss genes were found, PpSKI1 and PpSKI2, which encode highly similar proteins with homologues in vascular plants. Fusions of the two encoded proteins to the green fluorescent protein localize to the nucleus. Knockout mutants for either gene have an excess of gametophores under low light conditions, and exhibit reduced gametophore stem lengths. Possible functions of the new proteins and their connection to the SnRK1 kinase are discussed.

Effect of the energy supply on filamentous growth and development in Physcomitrella patens.

The filamentous gametophyte of the moss Physcomitrella patens consists of two filament types called chloronemata and caulonemata. Chloronemal cells are photosynthetically active with numerous chloroplasts, while caulonemata help to spread the colony by radial growth. The balance between the two filament types is affected by external factors such as light and plant hormones. In the present study, caulonema formation and chloronemal branching have been monitored during high and low light conditions and in the presence of glucose, auxin, or cytokinin. These experiments were performed both in a wild-type strain and in a hxk1 knockout mutant which lacks the major hexokinase of Physcomitrella. It was found that caulonema formation is induced by high energy conditions such as high light and external glucose, while chloronemal branching is stimulated by low energy conditions such as reduced light, and in the hxk1 mutant. The hxk1 mutation also causes buds to appear on chloronemal filaments, which is rarely seen in the wild type, and shows increased sensitivity to cytokinin and abscisic acid. Based on these findings a model is proposed in which the energy supply of the moss colony regulates the balance between chloronemal and caulonemal growth.

A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens.

Hexokinase catalyzes the first step in the metabolism of glucose but has also been proposed to be involved in sugar sensing and signaling both in yeast and in plants. We have cloned a hexokinase gene, PpHXK1, in the moss Physcomitrella patens where gene function can be studied directly by gene targeting. PpHxk1 is a novel type of chloroplast stromal hexokinase that differs from previously studied membrane-bound plant hexokinases. Enzyme assays on a knock-out mutant revealed that PpHxk1 is the major glucose-phosphorylating enzyme in Physcomitrella, accounting for 80% of the total activity in protonemal tissue. The mutant is deficient in the response to glucose, which in wild type moss induces the formation of caulonemal filaments that protrude from the edge of the colony. Growth on glucose in the dark is strongly reduced in the mutant. Sequence data suggest that most plants including Physcomitrella and Arabidopsis have both chloroplast-imported hexokinases similar to PpHxk1 and traditional membrane-bound hexokinases. We propose that the two types of plant hexokinases have distinct physiological roles.