Biomedical subjects

Göran Sandberg

Publications and source records attributed to Göran Sandberg.

At least 19 recordsLinked to original sources

PubMed2006

Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis.

Cytokinins, which are central regulators of cell division and differentiation in plants, are adenine derivatives carrying an isopentenyl side chain that may be hydroxylated. Plants have two classes of isopentenyltransferases (IPTs) acting on the adenine moiety: ATP/ADP isopentenyltransferases (in Arabidopsis thaliana, AtIPT1, 3, 4-8) and tRNA IPTs (in Arabidopsis, AtIPT2 and 9). ATP/ADP IPTs are likely to be responsible for the bulk of cytokinin synthesis, whereas it is thought that cis-zeatin (cZ)-type cytokinins are produced possibly by degradation of cis-hydroxy isopentenyl tRNAs, which are formed by tRNA IPTs. However, these routes are largely hypothetical because of lack of in vivo evidence, because the critical experiment necessary to verify these routes, namely the production and analysis of mutants lacking AtIPTs, has not yet been described. We isolated null mutants for all members of the ATP/ADP IPT and tRNA IPT gene families in Arabidopsis. Notably, our work demonstrates that the atipt1 3 5 7 quadruple mutant possesses severely decreased levels of isopentenyladenine and trans-zeatin (tZ), and their corresponding ribosides, ribotides, and glucosides, and is retarded in its growth. In contrast, these mutants possessed increased levels of cZ-type cytokinins. The atipt2 9 double mutant, on the other hand, lacked isopentenyl- and cis-hydroxy isopentenyl-tRNA, and cZ-type cytokinins. These results indicate that whereas ATP/ADP IPTs are responsible for the bulk of isopentenyladenine- and tZ-type cytokinin synthesis, tRNA IPTs are required for cZ-type cytokinin production. This work clarifies the long-standing questions of the biosynthetic routes for isopentenyladenine-, tZ-, and cZ-type cytokinin production.

Adenosine Diphosphate↗

PubMed2006

STY1 regulates auxin homeostasis and affects apical-basal patterning of the Arabidopsis gynoecium.

Gynoecia of the Arabidopsis mutant sty1-1 display abnormal style morphology and altered vascular patterning. These phenotypes, which are enhanced in the sty1-1 sty2-1 double mutant, suggest that auxin homeostasis or signalling might be affected by mutations in STY1 and STY2, both members of the SHI gene family. Chemical inhibition of polar auxin transport (PAT) severely affects the apical-basal patterning of the gynoecium, as do mutations in the auxin transport/signalling genes PIN1, PID and ETT. Here we show that the apical-basal patterning of sty1-1 and sty1-1 sty2-1 gynoecia is hypersensitive to reductions in PAT, and that sty1-1 enhances the PAT inhibition-like phenotypes of pin1-5, pid-8 and ett-1 gynoecia. Furthermore, we show that STY1 activates transcription of the flavin monooxygenase-encoding gene THREAD/YUCCA4, involved in auxin biosynthesis, and that changes in expression of STY1 and related genes lead to altered auxin homeostasis. Our results suggest that STY1 and related genes promote normal development of the style and affect apical-basal patterning of the gynoecium through regulation of auxin homeostasis.

Arabidopsis↗

PubMed2005

Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development.

A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.

Adaptation, Physiological↗

PubMed2005

Maintenance of embryonic auxin distribution for apical-basal patterning by PIN-FORMED-dependent auxin transport in Arabidopsis.

Molecular mechanisms of pattern formation in the plant embryo are not well understood. Recent molecular and cellular studies, in conjunction with earlier microsurgical, physiological, and genetic work, are now starting to define the outlines of a model where gradients of the signaling molecule auxin play a central role in embryo patterning. It is relatively clear how these gradients are established and interpreted, but how they are maintained is still unresolved. Here, we have studied the contributions of auxin biosynthesis, conjugation, and transport pathways to the maintenance of embryonic auxin gradients. Auxin homeostasis in the embryo was manipulated by region-specific conditional expression of indoleacetic acid-tryptophan monooxygenase or indoleacetic acid-lysine synthetase, bacterial enzymes for auxin biosynthesis or conjugation. Neither manipulation of auxin biosynthesis nor of auxin conjugation interfered with auxin gradients and patterning in the embryo. This result suggests a compensatory mechanism for buffering auxin gradients in the embryo. Chemical and genetic inhibition revealed that auxin transport activity, in particular that of the PIN-FORMED1 (PIN1) and PIN4 proteins, is a major factor in the maintenance of these gradients.

Arabidopsis↗

PubMed2005

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1.

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.

Arabidopsis↗

PubMed2005

Resistance of flight feathers to mechanical fatigue covaries with moult strategy in two warbler species.

Flight feather moult in small passerines is realized in several ways. Some species moult once after breeding or once on their wintering grounds; others even moult twice. The adaptive significance of this diversity is still largely unknown. We compared the resistance to mechanical fatigue of flight feathers from the chiffchaff Phylloscopus collybita, a migratory species moulting once on its breeding grounds, with feathers from the willow warbler Phylloscopus trochilus, a migratory species moulting in both its breeding and wintering grounds. We found that flight feathers of willow warblers, which have a shaft with a comparatively large diameter, become fatigued much faster than feathers of chiffchaffs under an artificial cyclic bending regime. We propose that willow warblers may strengthen their flight feathers by increasing the diameter of the shaft, which may lead to a more rapid accumulation of damage in willow warblers than in chiffchaffs.

Animal Migration↗

PubMed2005

Sites and regulation of auxin biosynthesis in Arabidopsis roots.

Auxin has been shown to be important for many aspects of root development, including initiation and emergence of lateral roots, patterning of the root apical meristem, gravitropism, and root elongation. Auxin biosynthesis occurs in both aerial portions of the plant and in roots; thus, the auxin required for root development could come from either source, or both. To monitor putative internal sites of auxin synthesis in the root, a method for measuring indole-3-acetic acid (IAA) biosynthesis with tissue resolution was developed. We monitored IAA synthesis in 0.5- to 2-mm sections of Arabidopsis thaliana roots and were able to identify an important auxin source in the meristematic region of the primary root tip as well as in the tips of emerged lateral roots. Lower but significant synthesis capacity was observed in tissues upward from the tip, showing that the root contains multiple auxin sources. Root-localized IAA synthesis was diminished in a cyp79B2 cyp79B3 double knockout, suggesting an important role for Trp-dependent IAA synthesis pathways in the root. We present a model for how the primary root is supplied with auxin during early seedling development.

Arabidopsis↗

PubMed2005

Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen.

Wood formation is a fundamental biological process with significant economic interest. While lignin biosynthesis is currently relatively well understood, the pathways leading to the synthesis of the key structural carbohydrates in wood fibers remain obscure. We have used a functional genomics approach to identify enzymes involved in carbohydrate biosynthesis and remodeling during xylem development in the hybrid aspen Populus tremula x tremuloides. Microarrays containing cDNA clones from different tissue-specific libraries were hybridized with probes obtained from narrow tissue sections prepared by cryosectioning of the developing xylem. Bioinformatic analyses using the sensitive tools developed for carbohydrate-active enzymes allowed the identification of 25 xylem-specific glycosyltransferases belonging to the Carbohydrate-Active EnZYme families GT2, GT8, GT14, GT31, GT43, GT47, and GT61 and nine glycosidases (or transglycosidases) belonging to the Carbohydrate-Active EnZYme families GH9, GH10, GH16, GH17, GH19, GH28, GH35, and GH51. While no genes encoding either polysaccharide lyases or carbohydrate esterases were found among the secondary wall-specific genes, one putative O-acetyltransferase was identified. These wood-specific enzyme genes constitute a valuable resource for future development of engineered fibers with improved performance in different applications.

Cell Wall↗

PubMed2005

hca: an Arabidopsis mutant exhibiting unusual cambial activity and altered vascular patterning.

By screening a T-DNA population of Arabidopsis mutants for alterations in inflorescence stem vasculature, we have isolated a mutant with a dramatic increase in vascular tissue development, characterized by a continuous ring of xylem/phloem. This phenotype is the consequence of premature and numerous cambial cell divisions in both the fascicular and interfascicular regions that result in the loss of the alternate vascular bundle/fiber organization typically observed in Arabidopsis stems. The mutant was therefore designated high cambial activity (hca). The hca mutation also resulted in pleiotropic effects including stunting and a delay in developmental events such as flowering and senescence. The physiological characterization of hca seedlings in vitro revealed an altered auxin and cytokinin response and, most strikingly, an enhanced sensitivity to cytokinin. These results were substantiated by comparative microarray analysis between hca and wild-type plants. The genetic analysis of hca indicated that the mutant phenotype was not tagged by the T-DNA and that the hca mutation segregated as a single recessive locus, mapping to the long arm of chromosome 4. We propose that hca is involved in mechanisms controlling the arrangement of vascular bundles throughout the plant by regulating the auxin-cytokinin sensitivity of vascular cambial cells. Thus, the hca mutant is a useful model for examining the genetic and hormonal control of cambial growth and differentiation.

Arabidopsis↗

PubMed2004

A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux.

Polar transport-dependent local accumulation of auxin provides positional cues for multiple plant patterning processes. This directional auxin flow depends on the polar subcellular localization of the PIN auxin efflux regulators. Overexpression of the PINOID protein kinase induces a basal-to-apical shift in PIN localization, resulting in the loss of auxin gradients and strong defects in embryo and seedling roots. Conversely, pid loss of function induces an apical-to-basal shift in PIN1 polar targeting at the inflorescence apex, accompanied by defective organogenesis. Our results show that a PINOID-dependent binary switch controls PIN polarity and mediates changes in auxin flow to create local gradients for patterning processes.

Arabidopsis↗

PubMed2004

A Populus EST resource for plant functional genomics.

Trees present a life form of paramount importance for terrestrial ecosystems and human societies because of their ecological structure and physiological function and provision of energy and industrial materials. The genus Populus is the internationally accepted model for molecular tree biology. We have analyzed 102,019 Populus ESTs that clustered into 11,885 clusters and 12,759 singletons. We also provide >4,000 assembled full clone sequences to serve as a basis for the upcoming annotation of the Populus genome sequence. A public web-based EST database (POPULUSDB) provides digital expression profiles for 18 tissues that comprise the majority of differentiated organs. The coding content of Populus and Arabidopsis genomes shows very high similarity, indicating that differences between these annual and perennial angiosperm life forms result primarily from differences in gene regulation. The high similarity between Populus and Arabidopsis will allow studies of Populus to directly benefit from the detailed functional genomic information generated for Arabidopsis, enabling detailed insights into tree development and adaptation. These data will also valuable for functional genomic efforts in Arabidopsis.

Ecosystem↗

PubMed2004

A high-resolution transcript profile across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity.

Plant growth is the result of cell proliferation in meristems, which requires a careful balance between the formation of new tissue and the maintenance of a set of undifferentiated stem cells. Recent studies have provided important information on several genetic networks responsible for stem cell maintenance and regulation of cell differentiation in the apical meristems of shoots and roots. Nothing, however, is known about the regulatory networks in secondary meristems like the vascular cambium of trees. We have made use of the large size and highly regular layered organization of the cambial meristem to create a high-resolution transcriptional map covering 220 microm of the cambial region of aspen (Populus tremula). Clusters of differentially expressed genes revealed substantial differences in the transcriptomes of the six anatomically homogenous cell layers in the meristem zone. Based on transcriptional and anatomical data, we present a model for the position of the stem cells and the proliferating mother cells in the cambial zone. We also provide sets of marker genes for different stages of xylem and phloem differentiation and identify potential regulators of cambial meristem activity. Interestingly, analysis of known regulators of apical meristem development indicates substantial similarity in regulatory networks between primary and secondary meristems.

Cell Differentiation↗

PubMed2004

A family of auxin-conjugate hydrolases that contributes to free indole-3-acetic acid levels during Arabidopsis germination.

Auxins are hormones important for numerous processes throughout plant growth and development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. We examined amidohydrolase gene expression using northern and promoter-beta-glucuronidase analyses and found overlapping but distinct patterns of expression. To examine the in vivo importance of auxin-conjugate hydrolysis, we generated a triple hydrolase mutant, ilr1 iar3 ill2, which is deficient in three of these hydrolases. We compared root and hypocotyl growth of the single, double, and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAA-Phe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis.

Amidohydrolases↗

PubMed2004

Derivatization for LC-electrospray ionization-MS: a tool for improving reversed-phase separation and ESI responses of bases, ribosides, and intact nucleotides.

We have developed a method for analyzing polar compounds by reversed-phase LC-ESI-MS following esterification of the analytes' free hydroxyl groups with propionyl or benzoyl acid anhydride. The method was applied to members of the plant hormone group cytokinins, which includes adenine bases, ribosides/glycosides, and nucleotides substituted at N-6 with an isoprenoid side chain, spanning a wide range of polarity. It was also used to analyze other compounds of biological importance, e.g., the nucleotides AMP, ADP, and ATP. The formation of more hydrophobic derivatives had a significant impact on two aspects of the analysis. The retention on a reversed-phase material was greatly increased without the use of any acetate/formate buffer or ion pairing reagent, and the ESI response was enhanced, due to the higher surface activities of the derivatives. Detection limits of propionylated cytokinins were in the high-attomole to low-femtomole range, an improvement by factors of 10-100 compared to previously reported figures. Using an automated SPE-based purification method, 12 endogenous cytokinins were quantified in extracts from 20- to 100-mg samples of leaves (from the plant Arabidopsis thaliana) with high accuracy and precision. Furthermore, the chromatographic properties of the benzoylated AMP, ADP, and ATP in the reversed-phase LC-MS system were much better in terms of retention, separation, and sensitivity than those of their underivatized counterparts, even without the use of any ion pairing reagent. Our data show that derivatization followed by LC-ESI-MS is an effective strategy for analyzing low molecular weight compounds, enabling compounds with a wide range of polarity to be determined in a single-injection LC-MS analysis.

Adenine↗

PubMed2004

Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana: a factor of potential importance for auxin-cytokinin-regulated development.

One of the most long-lived models in plant science is the belief that the long-distance transport and ratio of two plant hormones, auxin and cytokinin, at the site of action control major developmental events such as apical dominance. We have used in vivo deuterium labeling and mass spectrometry to investigate the dynamics of homeostatic cross talk between the two plant hormones. Interestingly, auxin mediates a very rapid negative control of the cytokinin pool by mainly suppressing the biosynthesis via the isopentenyladenosine-5'-monophosphate-independent pathway. In contrast, the effect of cytokinin overproduction on the entire auxin pool in the plant was slower, indicating that this most likely is mediated through altered development. In addition, we were able to confirm that the lateral root meristems are likely to be the main sites of isopentenyladenosine-5'-monophosphate-dependent cytokinin synthesis, and that the aerial tissue of the plant surprisingly also was a significant source of cytokinin biosynthesis. Our demonstration of shoot-localized synthesis, together with data demonstrating that auxin imposes a very rapid regulation of cytokinin biosynthesis, illustrates that the two hormones can interact also on the metabolic level in controlling plant development, and that the aerial part of the plant has the capacity to synthesize its own cytokinin independent of long-range transport from the root system.

Adenosine Monophosphate↗

PubMed2004

A transcriptional timetable of autumn senescence.

BACKGROUND: We have developed genomic tools to allow the genus Populus (aspens and cottonwoods) to be exploited as a full-featured model for investigating fundamental aspects of tree biology. We have undertaken large-scale expressed sequence tag (EST) sequencing programs and created Populus microarrays with significant gene coverage. One of the important aspects of plant biology that cannot be studied in annual plants is the gene activity involved in the induction of autumn leaf senescence. RESULTS: On the basis of 36,354 Populus ESTs, obtained from seven cDNA libraries, we have created a DNA microarray consisting of 13,490 clones, spotted in duplicate. Of these clones, 12,376 (92%) were confirmed by resequencing and all sequences were annotated and functionally classified. Here we have used the microarray to study transcript abundance in leaves of a free-growing aspen tree (Populus tremula) in northern Sweden during natural autumn senescence. Of the 13,490 spotted clones, 3,792 represented genes with significant expression in all leaf samples from the seven studied dates. CONCLUSIONS: We observed a major shift in gene expression, coinciding with massive chlorophyll degradation, that reflected a shift from photosynthetic competence to energy generation by mitochondrial respiration, oxidation of fatty acids and nutrient mobilization. Autumn senescence had much in common with senescence in annual plants; for example many proteases were induced. We also found evidence for increased transcriptional activity before the appearance of visible signs of senescence, presumably preparing the leaf for degradation of its components.

Aging↗

PubMed2003

Gravity-regulated differential auxin transport from columella to lateral root cap cells.

Gravity-induced root curvature has long been considered to be regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients, and the transport mechanisms involved, remain to be identified. Here, we describe a GFP-based auxin biosensor to monitor auxin during Arabidopsis root gravitropism at cellular resolution. We identify elevated auxin levels at the root apex in columella cells, the site of gravity perception, and an asymmetric auxin flux from these cells to the lateral root cap (LRC) and toward the elongation zone after gravistimulation. We differentiate between an efflux-dependent lateral auxin transport from columella to LRC cells, and an efflux- and influx-dependent basipetal transport from the LRC to the elongation zone. We further demonstrate that endogenous gravitropic auxin gradients develop even in the presence of an exogenous source of auxin. Live-cell auxin imaging provides unprecedented insights into gravity-regulated auxin flux at cellular resolution, and strongly suggests that this flux is a prerequisite for root gravitropism.

Arabidopsis↗

PubMed2003

Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus x canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography/frit-fast atom bombardment mass spectrometry.

A search for naturally occurring aromatic cytokinins (ARCKs) in Arabidopsis thaliana plants and Populus x canadensis leaves led to the discovery of four new plant hormone substances: 6-(2-methoxybenzylamino)purine (ortho-methoxytopolin, MeoT), 6-(3-methoxybenzylamino)purine (meta-methoxytopolin, MemT) (Fig. 1) and their 9-beta-D-ribofuranosyl derivatives. These substances were identified by liquid chromatography electrospray ionization mass spectrometry [LC (+)ESI-MS] and capillary-liquid chromatography/frit-fast atom bombardment-mass spectrometry [CapLC/frit-FAB-MS] after pre-column derivatization. The chemical structures were subsequently confirmed by chemical synthesis. Because of lack of heavy labelled internal standards, the endogenous levels of methoxytopolins in A. thaliana plants, Populus x canadensis leaves and samples derived from cultures of Agrobacterium tumefaciens strain GV3101 were determined by enzyme-linked immunosorbent assay (ELISA) of HPLC-fractionated extracts. While the levels of MeoT, MemT and their ribosides in A. thaliana shoots and Populus x canadensis leaves were relatively low (approximately 0.25-10 pmol g-1 FW for MeoT and MemT, respectively), the A. tumefaciens strain produced up to 600 times more of the newly identified substances. Cytokinin activity of methoxytopolines was demonstrated in three bioassays testing their ability to stimulate tobacco callus growth, to delay chlorophyll degradation in excised wheat leaves, and to induce betacyanin synthesis in Amaranthus caudatus var. atropurpurea cotyledons. Notably, their anti-senescing activity in the wheat leaf assay exceeded that of BAP and Z by almost 200%. Methoxytopolins are proposed to be new members of the biologically active aromatic cytokinin family, which might have specific physiological functions.

Journal Article↗