Mattias Thelander

Snf1‐related protein kinase 1 is needed for growth in a normal day–night light cycle

The yeast Snf1 protein kinase and its animal homologue, the AMP‐activated protein kinase, play important roles in metabolic regulation, by serving as energy gauges that turn off energy‐consuming processes and mobilize energy reserves during low‐energy conditions. The closest homologue of these kinases in plants is Snf1‐related protein kinase 1 (SnRK1). We have cloned two SnRK1‐encoding genes, PpSNF1a and PpSNF1b, in the moss Physcomitrella patens, where gene function can be studied directly by gene targeting in the haploid gametophyte. A snf1a snf1b double knockout mutant is viable, but lacks all Snf1‐like protein kinase activity. The mutant has a complex phenotype that includes developmental abnormalities, premature senescence and altered sensitivities to plant hormones. Remarkably, the double knockout mutant also requires continuous light, and is unable to grow in a normal day–night light cycle. This suggests that SnRK1 is needed for metabolic changes that help the plant cope with the dark hours of the night.

The Arabidopsis thaliana STYLISH1 Protein Acts as a Transcriptional Activator Regulating Auxin Biosynthesis

Biosynthesis of the plant hormone auxin must be tightly controlled. This work shows that the STYLISH1 protein of the plant species Arabidopsis thaliana plays an important role in this process by directly binding to and activating at least one of the auxin biosynthesis genes. 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.

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.

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.

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.

Two S-adenosylmethionine synthetase-encoding genes differentially expressed during adventitious root development in Pinus contorta.

Two S-adenosylmethionine synthetase (SAMS) cDNAs, PcSAMS1 and PcSAMS2, have been identified in Pinus contorta. We found that the two genes are differentially expressed during root development. Thus, PcSAMS1 is preferentially expressed in roots and exhibits a specific expression pattern in the meristem at the onset of adventitious root development, whereas PcSAMS2 is expressed in roots as well as in shoots and is down-regulated during adventitious root formation. The expression of the two SAMS genes is different from the SAMS activity levels during adventitious root formation. We conclude that other SAMS genes that remain to be characterized may contribute to the observed SAMS activity, or that the activities of PcSAMS1 and PcSAMS2 are affected by post-transcriptional regulation. The deduced amino acid sequences of PcSAMS1 and PcSAMS2 are highly divergent, suggesting different functional roles. However, both carry the two perfectly conserved motifs that are common to all plant SAMS. At the protein level, PcSAMS2 shares about 90% identity to other isolated eukaryotic SAMS, while PcSAMS1 shares less than 50% identity with other plant SAMS. In a phylogenetic comparison, PcSAMS1 seems to have diverged significantly from all other SAMS genes. Nevertheless, PcSAMS1 was able to complement a Saccharomyces cerevisiae sam1 sam2 double mutant, indicating that it encodes a functional SAMS enzyme.

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

Reproductive organ development of the moss Physcomitrella patens is affected by two SHI/STY genes and the plant hormone auxin. 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 GmGH3pro:GUS and PpPINApro:GFP-GUS, and the auxin-conjugating transgene PpSHI2pro: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.

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.

Two novel types of hexokinases in the moss Physcomitrella patens

BackgroundHexokinase 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.ResultsWe 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.ConclusionsWe 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.

Cloning by pathway activation in yeast: identification of an Arabidopsis thaliana F-box protein that can turn on glucose repression

We describe a method for identifying signal transducing proteins from other organisms by their ability to turn on a signalling pathway when they are expressed at high level in yeast. The method was tested on a cDNA library from Arabidopsis thaliana, which was screened for clones that can activate glucose repression in the absence of glucose. Six clones were characterized. One of them codes for AtGRH1, a new F-box protein that shows similarity to GRR1, a yeast protein involved in glucose repression. The ability of AtGRH1 to activate glucose repression is dependent on the MIG1 repressor. Two-hybrid experiments revealed that AtGRH1 can interact with AtSKP1a and AtSKP1b, two recently identified SKP1 homologues in Arabidopsis. Other clones identified in the screen encode the transcription factor AtEBP, the 14-3-3 protein AtGF14 and two new proteins: AtMYR1 and AtPOZ1. None of these proteins turn on glucose repression. Instead, they illustrate various other ways by which foreign proteins can interfere with expression of a yeast gene. We conclude that our method worked as expected in at least one case, and that it could be applied to other signalling pathways that are conserved between yeast and higher eukaryotes.