Joakim Palovaara

Conifer WOX-related homeodomain transcription factors, developmental consideration and expression dynamic of WOX2 during Picea abies somatic embryogenesis

In angiosperms, the WOX family of transcription factors has important functions in meristem regulation and in control of the partitioning of developing embryos into functional domains. In this study, a putative WOX2 homologous gene was isolated from Picea abies, and its expression pattern during somatic embryo development was followed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We used strategies of both absolute and relative quantification of gene expression, and benefits and disadvantages of the two methods are presented and discussed. During embryogenesis, PaWOX2 expression was highest at the earliest stages of development, but low levels were also detected in seedling tissues. No PaWOX2 expression was detected in a non-embryogenic cell culture, indicating that PaWOX2 plays a fundamental role during early somatic embryo development, and can be used as a possible marker for embryogenic potential. Additional results show that conifers, like angiosperms, contain a large number of WOX-related genes, many of them expressed during embryo development. In phylogenetic analysis based on the deduced homeodomain of retrieved pine and spruce EST sequences, no conifer WUS homolog was found. Neither did we find any homeodomain to cluster with WOX5. Interestingly, a clade including only conifer sequences derived from various tissues was resolved as sister to a PhyscomitrellaWOX-like gene, suggestive of the early origin of this gene family. Our results thus provide basic information for further studies of the evolution of this gene family and of their function in relation to meristem dynamics and specification of stem cells in gymnosperms.

The polar auxin transport inhibitor NPA impairs embryo morphology and increases the expression of an auxin efflux facilitator protein PIN during Picea abies somatic embryo development.

Auxin and polar auxin transport have been implicated in controlling embryo patterning and development in angiosperms but less is known from the gymnosperms. The aims of this study were to determine at what stages of conifer embryo development auxin and polar auxin transport are the most important for normal development and to analyze the changes in embryos after treatment with the polar auxin inhibitor N-1-naphthylphthalamic acid (NPA). For these studies, somatic embryos of Norway spruce (Picea abies L. Karst) were used. Growth on medium containing NPA leads to the formation of embryos with poor shoot apical meristem (SAM) and fused cotyledons, and to a pin-formed phenotype of the regenerated plantlets. The effect of NPA on embryo morphology was most severe if embryos were transferred to NPA-containing medium immediately before cotyledon initiation and SAM specification. Indole-3-acetic acid (IAA) was identified by immunolocalization in developing embryos. The highest staining intensity was seen in early staged embryos and then decreased as the embryos matured. No clear IAA-maxima was seen, although the apical parts of embryos, particularly the protoderm, and the suspensor cells appear to accumulate more IAA, as reflected by the staining pattern. The NPA treatment also caused expanded procambium and a broader root apical meristem in embryos, and a significant increase in the expression of a PIN1-like gene. Taken together, our results show that, for proper cotyledon initiation, correct auxin transport is needed only during a short period at the transition stage of embryo development, probably involving PIN efflux proteins and that a common mechanism is behind proper cotyledon formation within the species of angiosperms and conifers, despite their cotyledon number which normally differs.

Stimulation of growth by proteorhodopsin phototrophy involves regulation of central metabolic pathways in marine planktonic bacteria

Significance Bacteria control biogeochemical cycles of elements and fluxes of energy in the ocean. Discovery of a membrane photoprotein widespread in marine bacteria—proteorhodopsin—expanded their potential importance for global energy budgets, providing a novel mechanism to harness light energy. Yet, how proteorhodopsin-derived energy is used for cell metabolism remains largely unexplored. We combined experiments in a model marine bacterium with gene expression analyses. Light-stimulated growth coincided with a shift in carbon acquisition pathways, with anaplerotic CO2 fixation providing up to one-third of the cell carbon. Exposure to light resulted in the up-regulation of several central metabolism genes, including the glyoxylate shunt. Thus, light provides proteorhodopsin-containing bacteria with wider means to adapt to environmental variability than previously recognized. Proteorhodopsin (PR) is present in half of surface ocean bacterioplankton, where its light-driven proton pumping provides energy to cells. Indeed, PR promotes growth or survival in different bacteria. However, the metabolic pathways mediating the light responses remain unknown. We analyzed growth of the PR-containing Dokdonia sp. MED134 (where light-stimulated growth had been found) in seawater with low concentrations of mixed [yeast extract and peptone (YEP)] or single (alanine, Ala) carbon compounds as models for rich and poor environments. We discovered changes in gene expression revealing a tightly regulated shift in central metabolic pathways between light and dark conditions. Bacteria showed relatively stronger light responses in Ala compared with YEP. Notably, carbon acquisition pathways shifted toward anaplerotic CO2 fixation in the light, contributing 31 ± 8% and 24 ± 6% of the carbon incorporated into biomass in Ala and YEP, respectively. Thus, MED134 was a facultative double mixotroph, i.e., photo- and chemotrophic for its energy source and using both bicarbonate and organic matter as carbon sources. Unexpectedly, relative expression of the glyoxylate shunt genes (isocitrate lyase and malate synthase) was >300-fold higher in the light—but only in Ala—contributing a more efficient use of carbon from organic compounds. We explored these findings in metagenomes and metatranscriptomes and observed similar prevalence of the glyoxylate shunt compared with PR genes and highest expression of the isocitrate lyase gene coinciding with highest solar irradiance. Thus, regulatory interactions between dissolved organic carbon quality and central metabolic pathways critically determine the fitness of surface ocean bacteria engaging in PR phototrophy.

Comparative expression pattern analysis of WUSCHEL- related homeobox 2 (WOX2) and WOX8⁄9 in developing seeds and somatic embryos of the gymnosperm Picea abies

• In seed plants, current knowledge concerning embryonic pattern formation by polar auxin transport (PAT) and WUSCHEL-related homeobox (WOX) gene activity is primarily derived from studies on angiosperms, while less is known about these processes in gymnosperms. In view of the differences in their embryogeny, and the fact that somatic embryogenesis is used for mass propagation of conifers, a better understanding of embryo development is vital. • The expression patterns of PaWOX2 and PaWOX8/9 were followed with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) during seed and somatic embryo development in Norway spruce (Picea abies), and in somatic embryos treated with the PAT inhibitor N-1-naphthylphthalamic acid (NPA). • Both PaWOX2 and PaWOX8/9 were highly expressed at the early growth stages of zygotic and somatic embryos, and shared a similar expression pattern over the entire embryo. At later embryo stages, high expression of PaWOX8/9 became restricted to cotyledon primordia, epidermis, procambium and root apical meristem (RAM), which became most evident in NPA-treated somatic embryos, while expression of PaWOX2 was much lower. • Our results suggest an ancestral role of WOX in seed plant embryo development, and strengthen the proposed connection between PAT, PIN-FORMED (PIN) and WOX in the regulation of embryo patterning in seed plants.

Regulation of proteorhodopsin gene expression by nutrient limitation in the marine bacterium Vibrio sp. AND4

Proteorhodopsin (PR), a ubiquitous membrane photoprotein in marine environments, acts as a light-driven proton pump and can provide energy for bacterial cellular metabolism. However, knowledge of factors that regulate PR gene expression in different bacteria remains strongly limited. Here, experiments with Vibrio sp. AND4 showed that PR phototrophy promoted survival only in cells from stationary phase and not in actively growing cells. PR gene expression was tightly regulated, with very low values in exponential phase, a pronounced peak at the exponential/stationary phase intersection, and a marked decline in stationary phase. Thus, PR gene expression at the entry into stationary phase preceded, and could therefore largely explain, the stationary phase light-induced survival response in AND4. Further experiments revealed nutrient limitation, not light exposure, regulated this differential PR expression. Screening of available marine vibrios showed that the PR gene, and thus the potential for PR phototrophy, is found in at least three different clusters in the genus Vibrio. In an ecological context, our findings suggest that some PR-containing bacteria adapted to the exploitation of nutrient-rich micro-environments rely on a phase of relatively slowly declining resources to mount a cellular response preparing them for adverse conditions dispersed in the water column.

Expression of a gymnosperm PIN homologous gene correlates with auxin immunolocalization pattern at cotyledon formation and in demarcation of the procambium during Picea abies somatic embryo development and in seedling tissues

In seed plants, the body organization is established during embryogenesis and is uniform across gymnosperms and angiosperms, despite differences during early embryogeny. Evidence from angiosperms implicates the plant hormone auxin and its polar transport, mainly established by the PIN family of auxin efflux transporters, in the patterning of embryos. Here, PaPIN1 from Norway spruce (Picea abies [L.] Karst.), a gene widely expressed in conifer tissues and organs, was characterized and its expression and localization patterns were determined with reverse transcription polymerase chain reaction and in situ hybridization during somatic embryo development and in seedlings. PaPIN1 shares the predicted structure of other PIN proteins, but its central hydrophilic loop is longer than most PINs. In phylogenetic analyses, PaPIN1 clusters with Arabidopsis thaliana (L.) Heynh. PIN3, PIN4 and PIN7, but its expression pattern also suggests similarity to PIN1. The PaPIN1 expression signal was high in the protoderm of pre-cotyledonary embryos, but not if embryos were pre-treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This, together with a high auxin immunolocalization signal in this cell layer, suggests a role of PaPIN1 during cotyledon formation. At later stages, high PaPIN1 expression was observed in differentiating procambium, running from the tip of incipient cotyledons down through the embryo axis and to the root apical meristem (RAM), although the mode of RAM specification in conifer embryos differs from that of most angiosperms. Also, the PaPIN1 in situ signal was high in seedling root tips including root cap columella cells. The results thus suggest that PaPIN1 provides an ancient function associated with auxin transport and embryo pattern formation prior to the separation of angiosperms and gymnosperms, in spite of some morphological differences.

Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions

To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 106 cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 104 cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 106 bacteria per ml per day) and bacterial production (from 3 to 379 μg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.

WOX2 and polar auxin transport during spruce embryo pattern formation

The WOX family of transcription factors and polar auxin transport (PAT) are both essential for embryonic patterning and thus normal embryo development in angiosperms. Recent analysis by us of WOX-related genes in Picea and Pinus suggests that they play fundamental roles during embryo development also in conifers.1 It has been proposed that there is a connection between the spatial separation of WOX2 and WOX8, and PAT in the formation of the apical-basal axis in Arabidopsis embryos and that both are involved in the regulation of the auxin efflux carrier PIN1. Auxin also seems to play a crucial role in apical-basal axis formation in conifer embryos based on studies using the polar auxin inhibitor NPA. We recently analyzed the expression of a PIN1-like gene in NPA-treated and untreated precotyledonary somatic spruce embryos and could see a significant upregulation of the PIN1-like gene in the NPA-treated embryos.2 Here we show that PaWOX2 is also significantly upregulated in the same embryos. Taken together, this suggests that PAT is involved in regulating both PIN1 and WOX2 expression in conifers and strengthens the evidence for the proposed connection between WOX and PIN genes in seed plants.

Prokaryotic Responses to Ammonium and Organic Carbon Reveal Alternative CO2 Fixation Pathways and Importance of Alkaline Phosphatase in the Mesopelagic North Atlantic

To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC) fixation, community composition (16S rRNA sequencing) and community gene expression (metatranscriptomics) in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e., pyruvate plus acetate) were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates—assumed to be related to autotrophic metabolisms—were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention.