Urban trees and atmospheric pollutants in big cities: Effects in Madrid

Una investigación sobre la mejora de la contaminación del aire (CA) por medio de arbolado urbano se realizó en Madrid, una ciudad con casi 4 M de habitantes, 2,8 M de vehículos y casi 3 M de árboles de mantenimiento público. La mayoría de los árboles estaban en dos bosques periurbanos. Los 650.000 restantes era pies de alineación y parques. Los taxones estudiados fueron Platanus orientalis (97.205 árboles), Ulmus sp. (70.557), Pinus pinea (49.038), Aesculus hippocastanum (22.266), Cedrus sp. (13.678) y Quercus ilex (1.650), de calles y parques. Muestras foliares se analizaron en diferentes épocas del año, así como datos de contaminación por PM10 de 28 estaciones de medición de la contaminación durante 30 años, y también la intensidad del tráfico (IMD) en 2.660 calles. La acumulación de metales pesados (MP) sobre hojas y dentro de estas se estimó en relación con la CA y del suelo y la IMD del tráfico. La concentración media de Ba, Cd, Cr, Cu, Mn, Ni, Pb y Zn en suelo (materia seca) alcanzó: 489,5, 0,7, 49,4, 60,9, 460,9, 12,8, 155,9 y 190,3 mg kg-1 respectivamente. Los árboles urbanos, particularmente coníferas (debido a la mayor CA en invierno) contribuyen significativamente a mejorar la CA sobre todo en calles con alta IMD. La capacidad de las seis sp. para capturar partículas de polvo en su superficies foliares está relacionada con la IMD del tráfico y se estimó en 16,8 kg/año de MP tóxicos. Pb y Zn resultaron ser buenos marcadores antrópicos en la ciudad en relación con el tráfico, que fue la principal fuente de contaminación en los árboles y suelos de Madrid. Las especies de árboles variaron en función de su capacidad para capturar partículas (dependiendo de las propiedades de sus superficies foliares) y acumular los MP absorbidos de los suelos. Las concentraciones foliares de Pb y Zn estuvieron por encima de los límites establecidos en diferentes sitios de la ciudad. La microlocalización de Zn mediante microscópico mostró la translocación al xilema y floema. Se detectaron puntos de contaminación puntual de Cu and Cr en antiguos polígonos industriales y la distribución espacial de los MP en los suelos de Madrid mostró que en incluso en zonas interiores del El Retiro había ciertos niveles elevados de [Pb] en suelo, tal vez por el emplazamiento la Real Fábrica de Porcelana en la misma zona hace 200 años. Distintas áreas del centro de la ciudad también alcanzaron niveles altos de [Pb] en suelo. Según los resultados, el empleo de una combinación de Pinus pinea con un estrato intermedio de Ulmus sp. y Cedrus sp. puede ser la mejor recomendación como filtro verde eficiente. El efecto del ozono (O3) sobre el arbolado en Madrid fue también objeto de este estudio. A pesar de la reducción de precursores aplicada en muchos países industrializados, O3 sigue siendo la principal causa de CA en el hemisferio norte, con el aumento de [O3] de fondo. Las mayores [O3] se alcanzaron en regiones mediterráneas, donde el efecto sobre la vegetación natural es compensado por el xeromorfismo y la baja conductancia estomática en respuesta los episodios de sequía estival característicos de este clima. Durante una campaña de monitoreo, se identificaron daños abióticos en hojas de encina parecidos a los de O3 que estaban plantadas en una franja de césped con riego del centro de Madrid. Dada la poca evidencia disponible de los síntomas de O3 en frondosas perennifolias, se hizo un estudio que trató de 1) confirman el diagnóstico de daño de O3, 2) investigar el grado de los síntomas en encinas y 3) analizar los factores ambientales que contribuyeron a los daños por O3, en particular en lo relacionado con el riego. Se analizaron los marcadores macro y micromorfológicos de estrés por O3, utilizando las mencionadas encinas a modo de parcela experimental. Los síntomas consistieron en punteado intercostal del haz, que aumentó con la edad. Además de un punteado subyacente, donde las células superiores del mesófilo mostraron reacciones características de daños por O3. Las células próximas a las zonas dañadas, presentaron marcadores adicionales de estrés oxidativo. Estos marcadores morfológicos y micromorfológicos de estrés por O3 fueron similares a otras frondosas caducifolias con daños por O3. Sin embargo, en nuestro caso el punteado fue evidente con AOT40 de 21 ppm•h, asociada a riego. Análisis posteriores mostraron que los árboles con riego aumentaron su conductancia estomática, con aumento de senescencia, manteniéndose sin cambios sus características xeromórficas foliares. Estos hallazgos ponen de relieve el papel primordial de la disponibilidad de agua frente a las características xeromórficas a la hora de manifestarse los síntomas en las células por daños de O3 en encina. ABSTRACT Research about air pollution mitigation by urban trees was conducted in Madrid (Spain), a southern European city with almost 4 M inhabitants, 2.8 M daily vehicles and 3 M trees under public maintenance. Most trees were located in two urban forests, while 650'000 trees along urban streets and in parks. The urban taxa included Platanus orientalis (97'205 trees), Ulmus sp. (70’557), Pinus pinea (49'038), Aesculus hippocastanum (22’266), Cedrus sp. (13'678 and Quercus ilex (1'650) along streets and parks. Leave samples were analysed sequentially in different seasons, PM10 data from 28 air monitoring stations during 30 years and traffic density estimated from 2’660 streets. Heavy metal (HM) accumulation on the leaf surface and within leaves was estimated per tree related to air and soil pollution, and traffic intensity. Mean concentration of Ba, Cd, Cr, Cu, Mn, Ni, Pb and Zn in topsoil samples (dry mass) amounted in Madrid: 489.5, 0.7, 49.4, 60.9, 460.9, 12.8, 155.9 and 190.3 mg kg-1 respectively. Urban trees, particularly conifers (due to higher pollution in winter) contributed significantly to alleviate air pollution especially near to high ADT roads. The capacity of the six urban street trees species to capture air-born dust on the foliage surface as related to traffic intensity was estimated to 16.8 kg of noxious metals from exhausts per year. Pb and Zn pointed to be tracers of anthropic activity in the city with vehicle traffic as the main source of diffuse pollution on trees and soils. Tree species differed by their capacity to capture air-borne dust (by different leaf surface properties) and to allocate HM from soils. Pb and Zn concentrations in the foliage were above limits in different urban sites and microscopic Zn revelation showed translocation in xylem and phloem tissue. Punctual contamination in soils by Cu and Cr was identified in former industrial areas and spatial trace element mapping showed for central Retiro Park certain high values of [Pb] in soils even related to a Royal pottery 200 years ago. Different areas in the city centre also reached high levels [Pb] in soils. According to the results, a combination of Pinus pinea with understorey Ulmus sp. and Cedrus sp. layers can be recommended for the best air filter efficiency. The effects of ozone (O3) on trees in different areas of Madrid were also part of this study. Despite abatement programs of precursors implemented in many industrialized countries, ozone remained the main air pollutant throughout the northern hemisphere with background [O3] increasing. Some of the highest ozone concentrations were measured in regions with a Mediterranean climate but the effect on the natural vegetation is alleviated by low stomatal uptake and frequent leaf xeromorphy in response to summer drought episodes characteristic of this climate. During a bioindication survey, abiotic O3-like injury was identified in foliage. Trees were growing on an irrigated lawn strip in the centre of Madrid. Given the little structural evidence available for O3 symptoms in broadleaved evergreen species, a study was undertaken in 2007 with the following objectives 1) confirm the diagnosis, 2) investigate the extent of symptoms in holm oaks growing in Madrid and 3) analyse the environmental factors contributing to O3 injury, particularly, the site water supply. Therefore, macro- and micromorphological markers of O3 stress were analysed, using the aforementioned lawn strip as an intensive study site. Symptoms consisted of adaxial and intercostal stippling increasing with leaf age. Underlying stippling, cells in the upper mesophyll showed HR-like reactions typical of ozone stress. The surrounding cells showed further oxidative stress markers. These morphological and micromorphological markers of ozone stress were similar to those recorded in deciduous broadleaved species. However, stippling became obvious already at an AOT40 of 21 ppm•h and was primarily found at irrigated sites. Subsequent analyses showed that irrigated trees had their stomatal conductance increased and leaf life-span reduced whereas their leaf xeromorphy remained unchanged. These findings suggest a central role of water availability versus leaf xeromorphy for ozone symptom expression by cell injury in holm oak.

A non-persistently transmitted-virus induces a pull?push strategy inits aphid vector to optimize transmission and spread

Plant viruses are known to modify the behaviour of their insect vectors, both directly and indirectly,generally adapting to each type of virus?vector relationship in a way that enhances transmissionefficiency. Here, we report results of three different studies showing how a virus transmitted in a non-persistent (NP) manner (Cucumber mosaic virus; CMV, Cucumovirus) can induce changes in its host plant,cucumber (Cucumis sativus cv. Marumba) that modifies the behaviour of its aphid vector (Aphis gossypiiGlover; Hemiptera: Aphididae) in a way that enhances virus transmission and spread non-viruliferousaphids changed their alighting, settling and probing behaviour activities over time when exposed toCMV-infected and mock-inoculated cucumber plants. Aphids exhibited no preference to migrate fromCMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), butshowed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min afterrelease. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected overmock-inoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage andaphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph(EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of shortsuperficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (sec-ond hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much lesstime spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour includingan early increase in the number of short superficial probes and intracellular punctures followed by aphloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in aNP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting,settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spreadof the virus. Our findings should be considered when modelling the spread of viruses transmitted in a NPmanner.

Efecto de la infección por virus persistentes y no persistentes sobre el comportamiento alimenticio, la preferencia, tasa de aterrizaje y asentamiento y la eficacia biológica de Aphis gossypii Glover

En el complejo de plagas que atacan a los principales cultivos hortícolas protegidos, destacan principalmente los Hemípteros, y dentro de estos los pulgones, dada su importancia como vectores de virus que provocan considerables daños y pérdidas económicas. Debido a que la dispersión de la mayoría de los virus de plantas puede ser eficaz con densidades bajas de vectores y su control es muy complicado al no existir métodos curativos para su control, es necesario generar nuevos conocimientos sobre las interacciones virus-vector con el fin de desarrollar nuevas y eficaces estrategias de control. Por ello, el objetivo general de esta Tesis ha sido conocer el efecto de la infección viral (directo-mediado por la presencia del virus en el vector- e indirecto-mediado por las alteraciones físico-químicas que se originan en la planta como consecuencia de la infección viral-) sobre el comportamiento y eficacia biológica del vector Aphis gossypii Glover y sus posibles repercusiones en la epidemiología de virosis de transmisión no persistente (Cucumber mosaic virus, CMV, Cucumovirus) y persistente (Cucurbit aphid-borne yellows virus, CABYV, Polerovirus). El primer objetivo de esta Tesis Doctoral, se centró en el estudio del efecto indirecto del virus de transmisión no persistente CMV sobre el comportamiento alimenticio y la preferencia del pulgón A. gossypii en el cultivo de pepino. Los ensayos de despegue y aterrizaje mostraron que los pulgones que fueron liberados en las plantas de pepino infectadas con CMV tuvieron una mayor propensión en migrar hacia las plantas no infectadas (60, 120 y 180 minutos después de la liberación) que aquellos que fueron sometidos al tratamiento contrario (planta no infectada hacia planta infectada con CMV). El estudio de preferencia y asentamiento mostró que el vector A. gossypii prefiere asentarse en plantas infectadas con CMV en una etapa temprana de evaluación (30 minutos después de la liberación). Sin embargo, este comportamiento se revirtió en una etapa posterior (4 y 48 horas después de la liberación), donde los pulgones se asentaron más en las plantas no infectadas. A través de la técnica de Gráficos de Penetración Eléctrica (EPG) se observó un efecto indirecto del virus CMV, revelado por un cambio brusco en el comportamiento de prueba del pulgón a lo largo del tiempo, cuando éstos fueron expuestos a las plantas infectadas con CMV. Los primeros 15 minutos de registro EPG mostraron que los pulgones hicieron un número mayor de punciones intracelulares (potencial drops - pds) y pruebas en las plantas infectadas con CMV que en las plantas no infectadas. Por otra parte, la duración de la primera prueba fue más corta y la duración total de las pds por insecto fue mucho más larga en las plantas infectadas con CMV. Se observaron diferencias significativas en el tiempo transcurrido desde el final de la última pd hasta el final de la prueba, siendo ese tiempo más corto para los pulgones que estaban alimentándose en plantas infectadas con CMV. En la segunda hora de registro los pulgones rechazaron las plantas infectadas con CMV como fuente de alimento, permaneciendo menos tiempo en las fases de prueba en floema (fase de salivación – E1 y fase de ingestión del floema – E2). El comportamiento alimenticio observado sobre las plantas infectadas con CMV favorece la adquisición y posterior transmisión de los virus de transmisión no persistente, los cuales son adquiridos e inoculados durante la realización de pruebas intracelulares en las primeras pruebas de corta duración. En el segundo objetivo de la Tesis se evaluó el efecto directo e indirecto del virus de transmisión persistente CABYV en el comportamiento alimenticio y preferencia del pulgón A. gossypii en cultivo de pepino, especie susceptible al virus, y algodón, especie inmune al virus. No se observó un efecto directo del virus relevante en el comportamiento alimenticio del vector, ya que los resultados obtenidos a nivel floemático en plantas de pepino no se observaron en plantas de algodón, inmune al virus CABYV. Esto sugiere que los resultados obtenidos en pepino, pueden deberse a un “posible efecto indirecto” originado por la infección de las plantas susceptibles al virus durante la realización del ensayo, lo que indirectamente puede modificar el comportamiento del pulgón durante la fase de evaluación. Sin embargo, el virus CABYV modificó indirectamente el comportamiento alimenticio de su vector a través de cambios en la planta infectada. Los pulgones tardaron menos tiempo en llegar al floema, realizaron un mayor número de pruebas floemáticas y permanecieron durante más tiempo en actividades floemáticas en plantas infectadas con CABYV. El comportamiento observado sobre las plantas infectadas con CABYV favorece la adquisición de virus persistentes, los cuales son adquiridos durante la alimentación sostenida en floema. El estudio de preferencia y asentamiento de A. gossypii mostró que los pulgones virulíferos prefieren asentarse en plantas no infectadas a corto y largo plazo de evaluación (2, 4 y 48 horas después de la liberación). Los ensayos de despegue y aterrizaje mostraron que los pulgones virulíferos que fueron liberados en las plantas de pepino infectadas con CABYV tuvieron una mayor propensión en migrar hacia las plantas no infectadas (3, 6, 24 y 48 horas después de la liberación) que aquellos que fueron sometidos al tratamiento contrario (planta no infectada hacia planta infectada con CABYV). Sin embargo, los pulgones no virulíferos no mostraron preferencia por plantas de pepino no infectadas o infectadas con CABYV en ninguno de los ensayos (preferencia o despegue) o periodos evaluados (corto y largo plazo). Los resultados indican que el virus CABYV es capaz de modificar indirectamente el comportamiento alimenticio de su vector a través de cambios en la planta infectada, favoreciendo su adquisición por su principal vector, A. gossypii. Una vez que los pulgones tienen capacidad de transmitir el virus (virulíferos) se produce un cambio en su comportamiento prefiriendo asentarse sobre plantas no infectadas optimizándose así la dispersión viral. El tercer objetivo de la Tesis, fue evaluar los efectos directos e indirectos del virus CABYV así como los efectos indirectos del virus CMV en la eficacia biológica del vector A. gossypii. Los resultados obtenidos en los ensayos realizados con el virus persistente CABYV indican que el virus parece no modificar directamente ni indirectamente la eficacia biológica del vector en plantas de pepino o algodón, no observándose diferencias estadísticas en ninguno de los parámetros poblacionales evaluados (tiempo de desarrollo, tasa intrínseca de crecimiento, tiempo generacional medio, tasa media de crecimiento relativo y ninfas totales). En cuanto a los ensayos realizados con el virus no persistente, CMV, los resultados muestran un efecto indirecto del virus sobre la biología del vector. Así resultó que tanto la tasa intrínseca de crecimiento natural (rm) como la tasa media de crecimiento relativo (RGR) fueron más altas para pulgones crecidos sobre plantas infectadas con CMV que sobre plantas no infectadas, favoreciendo la reproducción y crecimiento poblacional del vector sobre plantas infectadas con CMV. Los resultados obtenidos en la presente Tesis, ofrecen un ejemplo de como los virus de plantas pueden manipular directa e indirectamente a su vector, maximizando así su dispersión entre las plantas. Esos nuevos conocimientos generados tienen implicaciones importantes en la transmisión, dispersión y en la epidemiología de los virus y deben ser considerados para diseñar o ajustar los modelos de simulación existentes y patrones de dispersión que describen las epidemias de estos virus. ABSTRACT The main objective of this Thesis has been to understand the effect of the viral infection (direct-mediated by the presence of the virus in the vector and indirect mediated by the chemical and physical changes originated in the plant as a consequence of the viral infection) on the behaviour and biological efficacy of the vector Aphis gossypii Glover and its consequences in the epidemiology of two viral diseases, one with non-persistent transmission (Cucumber mosaic virus, CMV, Cucumovirus) and another with persistent transmission (Cucurbit aphid-borne yellows virus, CABYV, Polerovirus). The first objective of this Thesis was the study of the indirect effect of the nonpersistent virus CMV on the feeding behaviour and preference of the aphid A. gossypii in cucumber plants. The results of the alighting and settling behaviour studies showed that aphids exhibited no preference to migrate from CMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), but showed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min after release. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected over mockinoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage and aphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph (EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of short superficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (second hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much less time spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour including an early increase in the number of short superficial probes and intracellular punctures followed by a phloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in a NP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting, settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spread of the virus. The second objective of this work was to evaluate the effects that the persistently aphid transmitted Cucurbit aphid-borne yellows virus (CABYV) can induce directly and indirectly on the alighting, settling and probing behaviour activities of the cotton aphid A. gossypii. Only minor direct changes on aphid feeding behaviour was observed due to CABYV when viruliferous aphids fed on mock-inoculated plants. However, the feeding behaviour of non-viruliferous aphids was very different on CABYV-infected than on mockinoculated plants. Non-viruliferous aphids spent longer time feeding from the phloem when plants were infected by CABYV than on mock-inoculated plants, suggesting that CABYV indirectly manipulates aphid feeding behaviour through its shared host plant in order to favour viral acquisition. The vector alighting and settling preference was compared between nonviruliferous and viruliferous aphids. Viruliferous aphids showed a clear preference for mockinoculated over CABYV-infected plants at short and long time, while such behaviour was not observed for non-viruliferous aphids. Overall, our results indicate that CABYV induces changes in its host plant that modifies aphid feeding behaviour in a way that virus acquisition from infected plants is enhanced. Once the aphids become viruliferous they prefer to settle on healthy plants, leading to optimize the transmission and spread of the virus. The third objective was to evaluate the direct and indirect effects of CABYV and indirect effects of the CMV on the A. gossypii fitness. Obtained results for the persistent virus CABYV showed that the virus did not modify the vector fitness in cucumber or cotton plants. None of the evaluated variables was statistically significant (development time (d), intrinsic growth rate (rm), mean relative growth rate (RGR) and total number of nymphs). On the other hand, data obtained for the non-persistent virus (CMV) showed an indirect effect of the virus on the vector fitness. Thus, the rm and RGR were higher for aphids grown on CMV-infected plants compared to aphids grown on mock-inoculated plants. Overall, the obtained results are clear examples of how plant viruses could manipulate directly and indirectly vector behaviour to optimize its own dispersion. These results are important for a better understanding of transmission, dispersion and epidemiology of plant viruses transmitted by vectors. This information could be also considered to design or adjust simulation models and dispersion patterns that describe plant virus epidemics.

Sucrose is a signal molecule in assimilate partitioning

The proton–sucrose symporter mediates the key transport step in the resource distribution system that allows many plants to function as multicellular organisms. In the results reported here, we identify sucrose as a signaling molecule in a previously undescribed signal-transduction pathway that regulates the symporter. Sucrose symporter activity declined in plasma membrane vesicles isolated from leaves fed exogenous sucrose via the xylem transpiration stream. Symporter activity dropped to 35–50% of water controls when the leaves were fed 100 mM sucrose and to 20–25% of controls with 250 mM sucrose. In contrast, alanine symporter and glucose transporter activities did not change in response to sucrose treatments. Decreased sucrose symporter activity was detectable after 8 h and reached a maximum by 24 h. Kinetic analysis of transport activity showed a decrease in Vmax. RNA gel blot analysis revealed a decrease in symporter message levels, suggesting a drop in transcriptional activity or a decrease in mRNA stability. Control experiments showed that these responses were not the result of changing osmotic conditions. Equal molar concentrations of hexoses did not elicit the response, and mannoheptulose, a hexokinase inhibitor, did not block the sucrose effect. These data are consistent with a sucrose-specific response pathway that is not mediated by hexokinase as the sugar sensor. Sucrose-dependent changes in the sucrose symporter were reversible, suggesting this sucrose-sensing pathway can modulate transport activity as a function of changing sucrose concentrations in the leaf. These results demonstrate the existence of a signaling pathway that can control assimilate partitioning at the level of phloem translocation.

Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors

In response to wounding, a 48-kDa myelin basic protein (MBP) kinase is activated within 2 min, both locally and systemically, in leaves of young tomato plants. The activating signal is able to pass through a steam girdle on the stem, indicating that it moves through the xylem and does not require intact phloem tissue. A 48-kDa MBP kinase is also activated by the 18-amino acid polypeptide systemin, a potent wound signal for the synthesis of systemic wound response proteins (swrps). The kinase activation by systemin is strongly inhibited by a systemin analog having a Thr-17 → Ala-17 substitution, which is a powerful antagonist of systemin activation of swrp genes. A 48-kDa MBP kinase activity also increases in response to polygalacturonic acid and chitosan but not in response to jasmonic acid or phytodienoic acid. In def1, a mutant tomato line having a defective octadecanoid pathway, the 48-kDa MBP kinase is activated by wounding and systemin as in the wild-type plants. This indicates that MBP kinase functions between the perception of primary signals and the DEF1 gene product. In response to wounding, the MBP kinase is phosphorylated on phosphotyrosine residues, indicating a relationship to the mitogen-activated protein kinase family of protein kinases.

Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola

Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids.

Nicotianamine Chelates Both FeIII and FeII. Implications for Metal Transport in Plants1

Nicotianamine (NA) occurs in all plants and chelates metal cations, including FeII, but reportedly not FeIII. However, a comparison of the FeII and ZnII affinity constants of NA and various FeIII-chelating aminocarboxylates suggested that NA should chelate FeIII. High-voltage electrophoresis of the FeNA complex formed in the presence of FeIII showed that the complex had a net charge of 0, consistent with the hexadentate chelation of FeIII. Measurement of the affinity constant for FeIII yielded a value of 1020.6, which is greater than that for the association of NA with FeII (1012.8). However, capillary electrophoresis showed that in the presence of FeII and FeIII, NA preferentially chelates FeII, indicating that the FeIINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H2O2-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2′-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2′-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed.

Manipulation of in Vivo Sorbitol Production Alters Boron Uptake and Transport in Tobacco1

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched 10B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of 10B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied 10B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.

Myo-Inositol-Dependent Sodium Uptake in Ice Plant1

In salt-stressed ice plants (Mesembryanthemum crystallinum), sodium accumulates to high concentrations in vacuoles, and polyols (myo-inositol, d-ononitol, and d-pinitol) accumulate in the cytosol. Polyol synthesis is regulated by NaCl and involves induction and repression of gene expression (D.E. Nelson, B. Shen, and H.J. Bohnert [1998] Plant Cell 10: 753–764). In the study reported here we found increased phloem transport of myo-inositol and reciprocal increased transport of sodium and inositol to leaves under stress. To determine the relationship between increased translocation and sodium uptake, we analyzed the effects of exogenous application of myo-inositol: The NaCl-inducible ice plant myo-inositol 1-phosphate synthase is repressed in roots, and sodium uptake from root to shoot increases without stimulating growth. Sodium uptake and transport through the xylem was coupled to a 10-fold increase of myo-inositol and ononitol in the xylem. Seedlings of the ice plant are not salt-tolerant, and yet the addition of exogenous myo-inositol conferred upon them patterns of gene expression and polyol accumulation observed in mature, salt-tolerant plants. Sodium uptake and transport through the xylem was enhanced in the presence of myo-inositol. The results indicate an interdependence of sodium uptake and alterations in the distribution of myo-inositol. We hypothesize that myo-inositol could serve not only as a substrate for the production of compatible solutes but also as a leaf-to-root signal that promotes sodium uptake.

Metabolism of d-Glycero-d-Manno-Heptitol, Volemitol, in Polyanthus. Discovery of a Novel Ketose Reductase1

Volemitol (d-glycero-d-manno-heptitol, α-sedoheptitol) is an unusual seven-carbon sugar alcohol that fulfills several important physiological functions in certain species of the genus Primula. Using the horticultural hybrid polyanthus (Primula × polyantha) as our model plant, we found that volemitol is the major nonstructural carbohydrate in leaves of all stages of development, with concentrations of up to 50 mg/g fresh weight in source leaves (about 25% of the dry weight), followed by sedoheptulose (d-altro-2-heptulose, 36 mg/g fresh weight), and sucrose (4 mg/g fresh weight). Volemitol was shown by the ethylenediaminetetraacetate-exudation technique to be a prominent phloem-mobile carbohydrate. It accounted for about 24% (mol/mol) of the phloem sap carbohydrates, surpassed only by sucrose (63%). Preliminary 14CO2 pulse-chase radiolabeling experiments showed that volemitol was a major photosynthetic product, preceded by the structurally related ketose sedoheptulose. Finally, we present evidence for a novel NADPH-dependent ketose reductase, tentatively called sedoheptulose reductase, in volemitol-containing Primula species, and propose it as responsible for the biosynthesis of volemitol in planta. Using enzyme extracts from polyanthus leaves, we determined that sedoheptulose reductase has a pH optimum between 7.0 and 8.0, a very high substrate specificity, and displays saturable concentration dependence for both sedoheptulose (apparent Km = 21 mm) and NADPH (apparent Km = 0.4 mm). Our results suggest that volemitol is important in certain Primula species as a photosynthetic product, phloem translocate, and storage carbohydrate.

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.

Transcription factor RF2a alters expression of the rice tungro bacilliform virus promoter in transgenic tobacco plants

The promoter from rice tungro bacilliform badnavirus (RTBV) is expressed only in phloem tissues in transgenic rice plants. RF2a, a b-Zip protein from rice, is known to bind to the Box II cis element near the TATA box of the promoter. Here, we report that the full-length RTBV promoter and a truncated fragment E of the promoter, comprising nucleotides −164 to +45, result in phloem-specific expression of β-glucuronidase (GUS) reporter genes in transgenic tobacco plants. When a fusion gene comprising the cauliflower mosaic virus 35S promoter and RF2a cDNA was coexpressed with the GUS reporter genes, GUS activity was increased by 2–20-fold. The increase in GUS activity was positively correlated with the amount of RF2a, and the expression pattern of the RTBV promoter was altered from phloem-specific to constitutive. Constitutive expression of RF2a did not induce morphological changes in the transgenic plants. In contrast, constitutive overexpression of the b-ZIP domain of RF2a had a strong effect on the development of transgenic plants. These studies suggest that expression of the b-Zip domain can interfere with the function of homologues of RF2a that regulate development of tobacco plants.

Characterization of Source- and Sink-Specific Sucrose/H+ Symporters from Carrot

To understand how sucrose (Suc) is transported from source leaves to developing tap roots of carrot (Daucus carota L.), we cloned two cDNAs (DcSUT1 and DcSUT2) for proteins with homologies to plant Suc/H+ symporters. The deduced polypeptide sequences are 52% identical and have 12 predicted membrane-spanning domains each. Transport activities were confirmed by expression of the clones in yeast cells. Both transporters had optimal activity below pH 5.0 and Michaelis constant values of 0.5 mm. Suc uptake was inhibited by protonophores, suggesting that Suc transport is linked to the proton electrochemical potential across the plasma membrane. DcSUT1 and DcSUT2 had markedly different expression patterns. Transcripts of DcSUT1 were found only in the green parts of plants, with highest levels in the lamina of source leaves, indicating that DcSUT1 is required for the loading of Suc into the phloem. In leaf lamina expression was diurnally regulated, suggesting that Suc export from the leaves is higher during the day than during the night. The mRNA of DcSUT2 was found mainly in sink organs, and no diurnal expression pattern was detected in the storage root. Here, expression was not restricted to the phloem but was much higher in storage parenchyma tissues of phloem and xylem. The close relationship of DcSUT2 with a Suc/H+ symporter from fava bean, which facilitates Suc uptake into the cotyledons of developing seeds, indicates that this carrot Suc transporter may be involved in loading Suc into storage parenchyma cells.

The H+-Sucrose Cotransporter NtSUT1 Is Essential for Sugar Export from Tobacco Leaves1

In many species translocation of sucrose from the mesophyll to the phloem is carrier mediated. A sucrose/H+-symporter cDNA, NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown to be highly expressed in mature leaves and at low levels in other tissues, including floral organs. To study the in vivo function of NtSUT1, tobacco plants were transformed with a SUT1 antisense construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly affected plants developed chloroses and necroses that led to death. The leaves exhibited impaired ability to export recently fixed 14CO2 and were unable to export transient starch during extended periods of darkness. As a consequence, soluble carbohydrates accumulated and photosynthesis was reduced. Autoradiographs of leaves show a heterogenous pattern of CO2 fixation even after a 24-h chase. The 14C pattern does not change with time, suggesting that movement of photosynthate between mesophyll cells may also be impaired. The affected lines show a reduction in the development of the root system and delayed or impaired flowering. Taken together, the effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic route and possibly for intermesophyll transport as well.

Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine Decarboxylase Promoters from Opium Poppy Are Conserved in Transgenic Tobacco1

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.