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Conifers are of great economic and ecological importance, but little is known concerning their genomic organization. This study is an attempt to obtain high-quality high-molecular-weight DNA from Pinus pinaster cotyledons and the construction of a pine BAC library. The preparation incorporates modifications like low centrifugation speeds, increase of EDTA concentration for plug maintenance, use of DNase inhibitors to reduce DNA degradation, use of polyvinylpyrrolidone and ascorbate to avoid secondary metabolites, and a brief electrophoresis of the plugs prior to their use. A total of 72 192 clones with an average insert size of 107 kb, which represents an equivalent of 11X pine haploid genomes, were obtained. The proportions of clones lacking inserts or containing chloroplast DNA are both approximately 1.6%. The library was screened with cDNA probes for seven genes, and two clones containing Fd-GOGAT sequences were found, one of them seemingly functional. Ongoing projects aimed at constructing a pinebacterial artificial chromosome library may benefit from the methods described here., QC 20120223

Lys-63-linked multiubiquitin chains play important roles in signal transduction in yeast and in mammals, but the functions for this type of chain in plants remain to be defined. The RING domain protein RGLG2 (for RING domain Ligase2) from Arabidopsis thaliana can be N-terminally myristoylated and localizes to the plasma membrane. It can form Lys-63-linked multiubiquitin chains in an in vitro reaction. RGLG2 has overlapping functions with its closest sequelog, RGLG1, and single mutants in either gene are inconspicuous. rglg1 rglg2 double mutant plants exhibit loss of apical dominance and altered phyllotaxy, two traits critically influenced by the plant hormone auxin. Auxin and cytokinin levels are changed, and the plants show a decreased response to exogenously added auxin. Changes in the abundance of PIN family auxin transport proteins and synthetic lethality with a mutation in the auxin transport regulator BIG suggest that the directional flow of auxin is modulated by RGLG activity. Modification of proteins by Lys-63-linked multiubiquitin chains is thus important for hormone-regulated, basic plant architecture.

Xylem is the plant vascular tissue responsible for raw sap conduction. It comprises two main types of cells that are derived from differentiating cambium: tracheary elements (TEs) and fibres that have conducting and mechanical role, respectively. Xylem formation is a developmental process and is under strict hormonal control involving a stew of phytohormones including auxin, cytokinin and ethylene.

A panel of aluminum-tolerant (AlRes) mutants was isolated by protoplast-based T-DNA activation tagging in the tobacco cultivar SR1. The mutants fell into two phenotypic classes: a minority of the mutants were fertile and developed similarly to the wild type (type I), the majority was male-sterile and grew as semi-dwarfs (type II). These traits, along with the aluminum tolerance, were inherited in a monogenic dominant manner. Both types of mutants were characterized by excessive bundling of actin microfilaments and by a strongly increased abundance of actin, a phenotype that could be partially phenocopied in the wild type by treatment with aluminum chloride. The actin bundles could be dissociated into finer strands by addition of exogenous auxin in both types of mutants. However, actin microfilaments and leaf expansion were sensitive to blockers of actin assembly in the wild type and in the mutants of type I, whereas they were more tolerant in the mutants of type II. The mutants of type II displayed a hypertrophic development of vasculature, manifest in form of supernumerary leaf veins and extended xylem layers in stems and petioles. Whereas mutants of type I were characterized by a normal, but aluminum-tolerant polar auxin-transport, auxin-transport was strongly promoted in the mutants of type II. The phenotype of these mutants is discussed in terms of reduced endocytosis leading, concomitantly with aluminum tolerance, to changes in polar auxin transport.

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS]A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development., © Springer Science+Business Media B.V. 2007

Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr.&Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar ( Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis ( Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants., QC 20100525

Stems and branches of angiosperm trees form tension wood (TW) when exposed to a gravitational stimulus. One of the main characteristics of TW, which distinguishes it from normal wood, is the formation of fibers with a thick inner gelatinous cell wall layer mainly composed of crystalline cellulose. Hence TW is enriched in cellulose, and deficient in lignin and hemicelluloses. An expressed sequence tag library made from TW-forming tissues in Populus tremula (L.) x tremuloides (Michx.) and data from transcript profiling using microarray and metabolite analysis were obtained during TW formation in Populus tremula (L.) in two growing seasons. The data were examined with the aim of identifying the genes responsible for the change in carbon (C) flow into various cell wall components, and the mechanisms important for the formation of the gelatinous cell wall layer (G-layer). A specific effort was made to identify carbohydrate-active enzymes with a putative function in cell wall biosynthesis. An increased C flux to cellulose was suggested by a higher abundance of sucrose synthase transcripts. However, genes related to the cellulose biosynthetic machinery were not generally affected, although the expression of secondary wall-specific CesA genes was modified in both directions. Other pathways for which the data suggested increased activity included lipid and glucosamine biosynthesis and the pectin degradation machinery. In addition, transcripts encoding fasciclin-like arabinogalactan proteins were particularly increased and found to lack true Arabidopsis orthologs. Major pathways for which the transcriptome and metabolome analysis suggested decreased activity were the pathway for C flux through guanosine 5'-diphosphate (GDP) sugars to mannans, the pentose phosphate pathway, lignin biosynthesis, and biosynthesis of cell wall matrix carbohydrates. Several differentially expressed auxin- and ethylene-related genes and transcription factors were also identified.

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., QC 20100525

A recent proteomic analysis of the thylakoid lumen of Arabidopsis thaliana revealed the presence of several PsbP-like proteins, and a homologue to this gene family was detected in the genome of the cyanobacterium Synechocystis sp. PCC 6803 (Schubert M, Petersson UA, Haas BJ, Funk C, Schroder WP, Kieselbach T (2002) J Biol Chem 277, 8354-8365). Using a peptide-directed antibody against this cyanobacterial PsbP-like protein (sll1418) we could show that it was localized in the thylakoid membrane and associated with Photosystem II. While salt washes did not remove the PsbP-like protein from the thylakoid membrane, it was partially lost during the detergent-based isolation of PSII membrane fractions. In total cell extracts this protein is present in the same amount as the extrinsic PsbO protein. We did not see any significant functional difference between the wild-type and a PsbP-like insertion mutant.

Photoinactivation of Photosystem II (PS II), the light-induced loss of ability to evolve oxygen, inevitably occurs under any light environment in nature, counteracted by repair. Under certain conditions, the extent of photoinactivation of PS II depends on the photon exposure (light dosage, x), rather than the irradiance or duration of illumination per se, thus obeying the law of reciprocity of irradiance and duration of illumination, namely, that equal photon exposure produces an equal effect. If the probability of photoinactivation (p) of PS II is directly proportional to an increment in photon exposure (p = kDeltax, where k is the probability per unit photon exposure), it can be deduced that the number of active PS II complexes decreases exponentially as a function of photon exposure: N = Noexp(-kx). Further, since a photon exposure is usually achieved by varying the illumination time (t) at constant irradiance (I), N = Noexp(-kI t), i.e., N decreases exponentially with time, with a rate coefficient of photoinactivation kI, where the product kI is obviously directly proportional to I. Given that N = Noexp(-kx), the quantum yield of photoinactivation of PS II can be defined as -dN/dx = kN, which varies with the number of active PS II complexes remaining. Typically, the quantum yield of photoinactivation of PS II is ca. 0.1micromol PS II per mol photons at low photon exposure when repair is inhibited. That is, when about 10(7) photons have been received by leaf tissue, one PS II complex is inactivated. Some species such as grapevine have a much lower quantum yield of photoinactivation of PS II, even at a chilling temperature. Examination of the longer-term time course of photoinactivation of PS II in capsicum leaves reveals that the decrease in N deviates from a single-exponential decay when the majority of the PS II complexes are inactivated in the absence of repair. This can be attributed to the formation of strong quenchers in severely-photoinactivated PS II comp

In this study, we have investigated the role of the CAO gene (coding for the chloroplast recognition particle cpSRP43) in the protection against and acclimation to environmental conditions that promote photooxidative stress. Deficiency of cpSRP43 in the Arabidopsis mutant chaos has been shown previously to lead to partial loss of a number of proteins of the photosystem II (PSII) antennae. In addition, as reported here, mutant plants have lower growth rates and reduced lignin contents under laboratory conditions. However, chaos seedlings showed significantly higher tolerance to photooxidative stress under both tightly controlled laboratory conditions and highly variable conditions in the field. This greater tolerance of chaos plants was manifested in less photooxidative damage together with faster growth recovery in young seedlings. It was also associated with a lower production of H2O2, lower ascorbate levels and less induction of ascorbate peroxidases. Under field conditions, chaos exhibited better overall photosynthetic performance and had higher survival rates. Expression of the CAO gene may be regulated by a light-dependent chloroplastic redox signalling pathway, and was inhibited during acclimation to high light and chilling temperatures, simultaneously with induction of ascorbate peroxidases. It is concluded that the presence/absence of the CAO gene has an impact on photo-produced H2O2, lignification in the hypocotyls and on the plant's susceptibility to photooxidative stress. Therefore, regulation of the CAO gene may be part of the plant's system for acclimation to high light and chilling temperatures.

The establishment of the dormant state in meristems involves considerable physiological and metabolic alterations necessary for surviving unfavourable growth conditions. However, a global molecular analysis of dormancy in meristems has been hampered by the difficulty in isolating meristem cells. We used cryosectioning to isolate purified cambial meristem cells from the woody plant Populus tremula during active growth and dormancy. These samples were used to generate meristem-specific cDNA libraries and for cDNA microarray experiments to define the global transcriptional changes underlying cambial dormancy. The results indicate a significant reduction in the complexity of the cambial transcriptome in the dormant state. Although cell division is terminated in the dormant cambium, the cell cycle machinery appears to be maintained in a skeletal state as suggested by the continued presence of transcripts for several cell cycle regulators. The downregulation of PttPIN1 and PttPIN2 transcripts explains the reduced basipetal polar auxin transport during dormancy. The induction of a member of the SINA family of ubiquitin ligases implicated in auxin signalling indicates a potential mechanism for modulation of auxin sensitivity during cambial dormancy. The metabolic alterations during dormancy are mirrored in the induction of genes involved in starch breakdown and the glyoxysomal cycle. Interestingly, the induction of RGA1 like gene suggests modification of gibberellin signalling in cambial dormancy. The induction of genes such as poplar orthologues of FIE and HAP2 indicates a potential role for these global regulators of transcription in orchestrating extensive changes in gene expression during dormancy.

NR 20160307

NR 2016

NR 2016

A culture protocol has been developed for mesophyll protoplasts isolated from various d¡haploid clones of potato. A large number of call¡ was obtained after serial dilution of the cultures w¡th a suitable amount and type of medium at different stages of cell colony development. A polyelhylene glycol fusion procedure was developed that yielded up to 12 % heterokaryons. Using this fusion protocol hybrid cells have been manually lsolated, cultured and regenerated into plants. Fus¡on products were identified 2-3 days after fuslon by the dual fluorescence emission from the chloroplasts in mesophyll protoplasts (red) and the fluorescein diacetate stain in l¡ght and norflurazon bleached protoplasts (yellow-green). This fusion and select¡on strategy leads to the recovery of almost 100 % hybr¡d plants as established by isozyme analysls. Cytolog¡cal analysis of protoplast-derived plants and somatic hybrid plants revealed genetic changes as a consequence of protoplast culture and protoplast fus¡on. Twenty-three tetraplo¡d somatic hybrid plants were obta¡ned from 6 different calli and 9 of these were euploid. Morphological assessments of somatic hybr¡d plants of different ploidy levels demonstrated that tetraploid as well as several hexaploid somatic hybrids showed an increased vigour as compared to the parental plants. Most tetraploid somat¡c hybrids had a similar appearance although not all euploid plants were identical. Loss of vigour was evident in all mixoploid and octoplo¡d plants. These were stunted, weak and had an abnormal leaf morphology. The plastid type in hybrid plants was determined by RFLP analysis. All analysed hybrids had a cpDNA restrict¡on fragment pattern ¡dent¡cal to one of the parents wh¡ch contained either S. tuberosum or S. stoloniferum cpDNA. A slight preference for S. tuberosum plastids was observed in hybild plants. No influence on tho assortment of chloroplast by the norflurazon bleaching could be detected., Financial support was given by the Swedish Council for Forestry and Agricultural Research.

A culture protocol has been developed for mesophyll protoplasts isolated from various dihaploid clones of potato. A special effort was made to promote the growth of initially dividing cells to form cell colonies and calli. An increase in plating efficiency in 3 different dihaploid clones and one doubled dihaploid clone was obtained after serial dilution of cultures with a suitable amount and type of medium at different stages of cell colony development. Plating on a refined semi-solid medium after 14 days of culture further improved both the yield and the quality of calli obtained. The refined plating medium also enhanced shoot regeneration ability from 67 to 90% in one of the dihaploid clones (67:9). The refined culture protocol could also be used without causing a decrease in plating efficiency at a low population density adjusted after 3 days of culture. The ploidy level of plants regenerated from dihaploid protoplasts were determined by chromosome counting and DNA analysis by flow cytometry. Most of the plants were aneuploid or tetraploid although, some dihaploid plants were obtained after protoplast culture of 2 dihaploid clones derived from the same cultivar (cv. Stina).

Green mesophyll protoplasts of the dihaploid potato line 198∶2 (Solanum tuberosum L.) were fused with herbicide-bleached mesophyll protoplasts of the dihaploid potato line 67∶9 using a polyethylene glycol protocol. Heterokaryons were identified under a fluorescence microscope using the dual fluorescence of carboxyfluorescein-stained, herbicide-bleached protoplasts and the autofluorescence of green mesophyll protoplasts. About 20% of the protoplasts survived the fusion treatment, and the fusion frequency was 3%-4%. Unfused and fused protoplasts were mass cultured for 6 weeks after which vigorously growing calli were selected and transferred to shoot regeneration medium. Somatic hybrids were identified by a combination of five isozyme markers, and the ploidy level was determined by flow cytometry. Out of 15 calli that regenerated shoots, 6 plants derived from 2 different calli were identified as hexaploid somatic hybrids, while one morphologically deviant plant from a third callus was identified as a mixoploid that had lost some enzyme markers after 4 months of culturing.

QC 20101108

QS 2015

In the genome of Arabidopsis thaliana five genes encode members of the FtsH (Filamentation temperature sensitive protein H) protease family with mutations or deletions in their proteolytic site. Despite of not being active proteases these so called FtsHi (i for inactive) enzymes still seem to be highly important for plant development. All five enzymes have been localized within the plant chloroplast, most of them seem to be inserted in the chloroplast envelope. As homozygous ftshi mutants are seed lethal, here we compared different heterozygous ftshi mutants with respect to their growth at various light periods and at semi-natural growth conditions in the field. Their photosynthetic efficiency was evaluated by pulse-amplitude modulated fluorescence and the composition of their photosynthetic complexes was analysed by native polyacrylamide gel electrophoresis. Co-expression analyses were performed to find clues about the function of the five FtsHi inactive proteases.

Two types of nitrogen-fixing root nodules are known, legume nodules and actinorhizal nodules. During the induction of nitrogen-fixing root nodules, phytohormones are involved in regulating gene expression to shape essential processes. One example is the expression of the early nodulin NODULE INCEPTION (NIN), which in the model legume Lotus japonicus is induced by cytokinin. In order to develop a bioassay for a quick and superficial analysis how phytohormones affect the expression of nodulation-related genes in different root nodule-forming plants, an axenic liquid culture system was established. The bioassay was used to examine responses in roots of the actinorhizal plant Datisca glomerata to phytohormones using quantitative RT-PCR. The synthetic cytokinin 6-Benzylaminopurine (BAP), the natural auxin phenyl acetic acid (PAA), and the synthetic auxin 1-Naphthaleneacetic Acid (NAA) were used. Marker genes for auxins and cytokinin responses were identified. PAA induced the expression of DgSAUR1, whereas NAA induced DgGH3.1. Induction of Lotus japonicus NIN was analysed for proof of concept, and the bioassay showed induction by cytokinin and auxin (NAA). D. glomerata NIN1 was induced by PAA, NAA, and more weakly by BAP. The gene encoding a transcription factor that activates NIN expression, CYCLOPS, was induced by PAA and NAA. To see whether induction of NIN1 expression led to the activation of target genes of NIN, orthologs of the transcription factors NF-YA1, NSP1 and NSP2, and of ERN1 were identified in D. glomerata and the transcription of the corresponding genes was analysed for regulation by BAP, PAA or NAA. Induction by PAA was found for all genes examined. Additionally, BAP and PAA were used individually or in combination with antagonists of ethylene or gibberellin biosynthesis, respectively. Both antagonists abolished the induction of NIN by BAP or PAA; furthermore, no induction of NIN was found when BAP and PAA were applied together. Altogether, these results

Two types of root nodule symbioses between nitrogen-fixing soil bacteria and higher plants are known, rhizobia/legume symbioses and the symbiosis between soil actinobacteria of the genus Frankia and more than 200 species from three different orders, collectively called actinorhizal plants. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen. Based on studies with bacterial mutants, it is known that rhizobia are supplied with dicarboxylates, specifically malate, but no legume malate exporters involved in the symbiosis have been identified thus far. For Frankia, a malate exporter from the NPF transporter family that locates to the perisymbiont membrane was identified in Alnus glutinosa (AgDCAT1; Jeong et al., 2004). In this study, the substrate specificity of two homologs of AgDCAT1 from nodules of two different actinorhizal hosts was analysed: CgDCAT1 from Casuarina glauca (Casuarinaceae, Fagales), a close relative of A. glutinosa, and DgDCAT1 from Datisca glomerata (Datiscaceae, Cucurbitales). A Multidrug And Toxic compound Extrusion (MATE) type protein from D. glomerata nodules (DgMATE1) was included in the analysis. The transcription of the corresponding genes of all three proteins was induced strongly in nodules compared to roots. Experiments using Xenopus laevis oocytes pre-loaded with 13C-labeled metabolites showed that all three proteins represent citrate exporters. Studies on the levels of citrate cycle intermediates showed that both citrate and malate were present at dramatically higher levels that 2‑oxoglutarate, succinate and fumate in roots and nodules of C. glauca as well as D. glomerata. The Casuarina-nodulating strain Frankia casuarinae CcI3 grew equally well on malate or citrate. In short, the results of this study led to the conclusion that intracellular Frankia bacteria are fed with citrate, not malate, in nodules of C. glauca and D. glomerata. AgDCAT1 and CgDCAT1 represent orthologs, but DgDCAT1 maps to another sub

Several types of cysteine-rich peptides are involved in the interaction between plants and microorganisms, e.g., anti-bacterial and anti-fungal defensins, characterized by eight conserved cysteine residues, or nodule-specific cysteine-rich peptides that manipulate microsymbiont development in legume nodules, containing five to six conserved cysteine residues. Nodule-specific defensins are found in all actinorhizal plants of Fagales, Rosales as well as Cucurbitales, and a defensin from Alnus glutinosa nodules was shown to affect membrane integrity of a compatible Frankia strain and cause leakage of amino acids (Carro et al., 2015). Nodules of Datisca glomerata (Datiscaceae. Cucurbitales) contain a gene family encoding nodule-specific defensins with an acidic C-terminal domain. The member expressed at the highest levels, DgDef1, was expressed in young infected cells and, transiently, above the nodule lobe meristem. Neither the complete protein excluding the N-terminal signal peptide produced in Pichia pastoris, nor the synthetically produced cysteine-rich domain had not effect on the growth of Gram-positive Streptomyces coelicolor. However, the cysteine-rich domain alone had a cytotoxic effect on Gram-negative E. coli K-12 and on Sinorhizobium meliloti 1021 with an ID50 of 20.8 µM. RNAseq analysis of Sm1021 cultures treated with this domain, compared to an untreated control, showed a strong effect on the expression of genes encoding transporters, e.g., a dicarboxylate uptake system, consistent with the fact that treated Sm1021 cells had leaky membrane as denoted by propidium iodide staining. However, the expression of the cell cycle regulator ctrA was not affected, suggesting that DgDef1 did not affect the ploidity of Sm1021. Phylogenetic analysis of the cysteine-rich domains showed that legume NCRs and actinorhizal nodule-specific defensins go back to a common ancestor.