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Physiological and Environmental Requirements for Poplar (Populus deltoides) Bark Storage Protein Degradation.

In poplar (Populus deltoides Bartr. ex Marsh), a 32-kD bark storage protein (BSP) accumulates in the bark during autumn and winter and declines during spring shoot growth. We investigated the physiological and environmental factors necessary for the degradation of poplar BSP. Poplar plants were exposed to short-day (SD) photoperiods for either 28 or 49 d. Plants exposed to short days for 28 d formed a terminal bud but were not dormant, whereas exposure to short days for 49 d induced bud dormancy. BSP accumulated in bark of plants exposed to both SD treatments. The level of BSP declined rapidly when nondormant plants were returned to long days. BSP levels did not decline in dormant plants that were exposed to long-day (LD) conditions. If dormant plants were first treated with either low temperatures (0[deg]C for 28 d) or with 0.5 M H2CN2 to overcome dormancy and then returned to long days, the level of BSP declined. Removal of buds from non-dormant or dormant plants in which dormancy had been overcome inhibited the degradation of BSP in LD conditions. BSP mRNA levels rapidly declined in plants exposed to long days, irrespective of the dormancy status of the plants or the presence or absence of buds. These results indicate that the buds of poplars are somehow able to communicate with bark storage sites and regulate poplar BSP degradation. These results further support an association of BSP mRNA levels with photoperiod because short days stimulate BSP mRNA accumulation, whereas long days result in a decline of BSP mRNA abundance.

Journal Article↗

Regulation of sulfur nutrition in wild-type and transgenic poplar over-expressing gamma-glutamylcysteine synthetase in the cytosol as affected by atmospheric H2S.

This study with poplar (Populus tremula x Populus alba) cuttings was aimed to test the hypothesis that sulfate uptake is regulated by demand-driven control and that this regulation is mediated by phloem-transported glutathione as a shoot-to-root signal. Therefore, sulfur nutrition was investigated at (a) enhanced sulfate demand in transgenic poplar over-expressing gamma-glutamylcysteine (gamma-EC) synthetase in the cytosol and (b) reduced sulfate demand during short-term exposure to H2S. H(2)S taken up by the leaves increased cysteine, gamma-EC, and glutathione concentrations in leaves, xylem sap, phloem exudate, and roots, both in wild-type and transgenic poplar. The observed reduced xylem loading of sulfate after H2S exposure of wild-type poplar could well be explained by a higher glutathione concentration in the phloem. In transgenic poplar increased concentrations of glutathione and gamma-EC were found not only in leaves, xylem sap, and roots but also in phloem exudate irrespective of H(2)S exposure. Despite enhanced phloem allocation of glutathione and its accumulation in the roots, sulfate uptake was strongly enhanced. This finding is contradictory to the hypothesis that glutathione allocated in the phloem reduces sulfate uptake and its transport to the shoot. Correlation analysis provided circumstantial evidence that the sulfate to glutathione ratio in the phloem may control sulfate uptake and loading into the xylem, both when the sulfate demand of the shoot is increased and when it is reduced.

Biological Transport↗

EST data suggest that poplar is an ancient polyploid.

We analysed the publicly available expressed sequence tag (EST) collections for the genus Populus to examine whether evidence can be found for large-scale gene-duplication events in the evolutionary past of this genus. The ESTs were clustered into unigenes for each poplar species examined. Gene families were constructed for all proteins deduced from these unigenes, and K(S) dating was performed on all paralogs within a gene family. The fraction of paralogs was then plotted against the K(S) values, which resulted in a distribution reflecting the age of duplicated genes in poplar. Sufficient EST data were available for seven different poplar species spanning four of the six sections of the genus Populus. For all these species, there was evidence that a large-scale gene-duplication event had occurred. From our analysis it is clear that all poplar species have shared the same large-scale gene-duplication event, suggesting that this event must have occurred in the ancestor of poplar, or at least very early in the evolution of the Populus genus.

Biological Evolution↗

Rhizosphere competitiveness of trichloroethylene-degrading, poplar-colonizing recombinant bacteria.

Indigenous bacteria from poplar tree (Populus canadensis var. eugenei 'Imperial Carolina') and southern California shrub rhizospheres, as well as two tree-colonizing Rhizobium strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 by integrating the tom locus into the chromosome. The poplar and Rhizobium recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 x 10(5) to 23 x 10(5) CFU/cm of root). In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% +/- 12% of all rhizosphere bacteria after 28 days (0.2 x 10(5) to 31 x 10(5) CFU/cm of root). Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as Pseudomonas sp. strain PsK recombinants) retained the ability to express TOM for 29 days as 100% +/- 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively.

Biodegradation, Environmental↗

[Analysis of salt resistance on the poplar transferred with salt tolerance gene].

Poplar(Pupulus x Xiao zhannica, cv. "balizhuang-yang") transferred with mtl-D gene was used as an experimental material. Through tissue culture, aquaculture and pot culture transgenic poplars are tested with variant Nacl salt gradient. In it transgenic poplar raises its initial days for differentiation, differentiation rate, bud intensity, bud height and growth potential than control plant. At the same time, the transgenic poplar has higher rooting rate and more top roots, side roots and higher root length than control under the same salt stress. The result shows that the transgenic poplar has better tolerance ability than control plant in the salt 4@1000 contensity mediums.

Adaptation, Physiological↗

[Introduction and selection of new poplar clones in shelterbelts/windbreaks in northern Liaoning].

Six new poplar clones were introduced in Qianshuanging township of Changtu county, Liaoning Province as experiment materials, and their indeces in growth, cold resistance, disease infection, and insect infection were evaluated and compared to the locally grown Fuzha II poplar. According to the comparison of the 4 indices, the study found that N-119 poplar showed all the scores obviously better than those of CK and other poplars, and was recommended for the area. The growth of Zhonglin 95 was higher, and its disease and pest infection was equal or near to the CK. So, the poplar Zhonglin 95 is also worthy to be recommended.

Agriculture↗

[Effects of 6-BA and AsA on photosynthesis photoinhibition of attached poplar leaves under osmotic stress of root].

In order to know more about the relationships between photosynthesis photoinhibition and reactive oxygen species metabolism, the effects of 6-benzyladenine (6-BA) and ascorbate (AsA) on net photosynthetic rate (Pn), apparent quantum yield (AQY), superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities, O2-* generation rate, and H2O2 and malondialdehyde (MDA) contents were studied with attached leaves of poplar clone seedlings under osmotic stress of root. Under osmotic stress, the photosynthesis photoinhibition of attached poplar leaves, judged by the significant decrease of Pn and AQY, was aggravated, and the balance of reactive oxygen species metabolism was destroyed. The superoxide dismutase (SOD) activity increased, but ascorbate peroxidase (APX) activity decreased. In the meantime, the O2-* generation rate and the contents of H2O2 and malondialdehyde (MDA) increased. When osmotic stressed poplar seedlings were pretreated with 6-BA and AsA, the activities of SOD and APX increased, O2-* generation rate and H2O2 and MDA contents decreased, and photosynthesis photoinhibition was alleviated. The contents of reactive oxygen species and MDA in poplar leaves were negatively correlated with net photosynthetic rate and apparent quantum yield. It's indicated that the photosynthesis photoinhibition of attached leaves of poplar clone seedlings had intrinsic relations with the accumulation of reactive oxygen species under osmotic stress of root, and the alleviation effects of 6-BA and AsA on photosynthesis photoinhibition were related to their promotion effects to the scavenging system of reactive oxygen species.

Ascorbic Acid↗

Cloning and sequencing of a cDNA for poplar endo-1,4-beta-glucanase.

A cDNA for poplar endo-1,4-beta-glucanase was cloned by use of a synthetic oligonucleotide as probe. The probe was designed on the basis of the N-terminal amino acid sequence of the beta-glucanase from suspension-cultured poplar cells (Populus alba L.), and by complete nucleotide sequence of the cDNA was determined. The 1,614-bp cDNA contained an open reading frame of 1,482 base pairs, encoding 494 amino acids. Removal of a putative signal sequence from the deduced amino acid sequence of the polypeptide yielded a mature protein of 467 amino acids. Comparison of deduced amino acid sequences revealed that the poplar endo-1,4-beta-glucanase was 80% and 70% identity to avocado fruit and bean abscission endo-1,4-beta-glucanases, respectively. Hydropathy plot analysis of the deduced amino acid sequence suggests that poplar endo-1,4-beta-glucanase belongs to family E in terms of the cellulase catalytic domain, and avocado fruit and abscission bean endo-1,4-beta-glucanases also belong to this family. 2,4-D markedly increased the level of the endo-1,4-beta-glucanase mRNA in cultured cells, while GA3, benzyladenine and abscisic acid each repressed transcription of this mRNA. The transcript was also detected in the roots and stems of intact plants, although the level of mRNA was much lower in intact tissues than in cultured cells. Genomic Southern analysis indicated that a small family of gene for endo-1,4-beta-glucanase exists in poplar.

Amino Acid Sequence↗

Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.

Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.

Populus↗

Bioconversion of hybrid poplar to ethanol and co-products using an organosolv fractionation process: optimization of process yields.

An organosolv process involving extraction with hot aqueous ethanol has been evaluated for bioconversion of hybrid poplar to ethanol. The process resulted in fractionation of poplar chips into a cellulose-rich solids fraction, an ethanol organosolv lignin (EOL) fraction, and a water-soluble fraction containing hemicellulosic sugars, sugar breakdown products, degraded lignin, and other components. The influence of four independent process variables (temperature, time, catalyst dose, and ethanol concentration) on product yields was analyzed over a broad range using a small composite design and response surface methodology. Center point conditions for the composite design (180 degrees C, 60 min, 1.25% H(2)SO(4), and 60% ethanol), yielded a solids fraction containing approximately 88% of the cellulose present in the untreated poplar. Approximately 82% of the total cellulose in the untreated poplar was recovered as monomeric glucose after hydrolysis of the solids fraction for 24 h using a low enzyme loading (20 filter paper units of cellulase/g cellulose); approximately 85% was recovered after 48 h hydrolysis. Total recovery of xylose (soluble and insoluble) was equivalent to approximately 72% of the xylose present in untreated wood. Approximately 74% of the lignin in untreated wood was recovered as EOL. Other cooking conditions resulted in either similar or inferior product yields although the distribution of components between the various fractions differed markedly. Data analysis generated regression models that describe process responses for any combination of the four variables.

Biodegradation, Environmental↗

Validated methods for the quantification of biologically active constituents of poplar-type propolis.

The validation of rapid, low-cost spectrophotometric procedures for the quantification of the three main groups of bioactive substances (flavones and flavonols, flavanones and dihydroflavonols, and total phenolics) in poplar-type propolis has been performed. A spectrophotometric assay based on the formation of an aluminium chloride complex was applied for the quantification of total flavones and flavonols using galangin as standard. Because of the high amount of flavanones and dihydroflavonols in "poplar type" propolis, the introduction of a distinct procedure for their quantification was considered of special significance and the DAB9 colorimetric method was applied for the purpose. Total phenolic content was measured by the Folin-Ciocalteu procedure using a mixture of pinocembrin and galangin as a reference. The procedures were validated using a model mixture of compounds representing the poplar-type propolis composition as found in previous studies. The accuracy (recovery) varied in the range 84-109%, and the relative standard deviation was 0.5-6.2%. The developed spectrophotometric procedures were applied to six poplar type propolis samples. The results were verified independently by a HPLC procedure. The two sets of results agreed satisfactory, as proven by Student's t-test.

Anti-Infective Agents↗

Mapping the proteome of poplar and application to the discovery of drought-stress responsive proteins.

Poplar is the first forest tree genome to be decoded. As an initial step to the comprehensive analysis of poplar proteome, we described reference 2-D-maps for eight tissues/organs of the plant, and the functional characterization of some proteins. A total of 398 proteins were excised from the gels. About 91.2% were identified by nanospray LC-MS/MS, based on comparison with 260,000 Populus sp. ESTs. In comparison, reliable PMFs were obtained for only 51% of the spots by MALDI-TOF-MS, from which 43% (83 spots) positively matched gene models of the Populus trichocarpa genome sequence. Among these 83 spots, 58% matched with the same proteins as identified by LC-MS/MS, 21.7% with unknown function proteins and 19.3% with completely different functions. In the second phase, we studied the effect of drought stress on poplar root and leaf proteomes. The function of up- and down-regulated proteins is discussed with respect to the physiological response of the plants and compared with transcriptomic data. Some important clues regarding the way poplar copes with water deficit were revealed.

Databases, Protein↗

Wound-induced and developmental activation of a poplar tree chitinase gene promoter in transgenic tobacco.

Wounding hybrid poplar (Populus trichocarpa x P. deltoides) trees results in the expression of novel wound-inducible (win) mRNAs thought to encode proteins involved in defense against pests and pathogens. Members of the win6 gene family encode acidic multi-domain chitinases, with combined structure and charge characteristics that differ from previously described chitinases. Win6 expression has been shown to occur in pooled unwounded leaves of a wounded (on multiple leaves) poplar plant. Here we demonstrate that wounding a single leaf induces win6 expression locally, in the wounded leaf, and remotely, in specific unwounded leaves with strong vascular connections to the wounded leaf. We also demonstrate that a win6 promoter-beta-glucuronidase (GUS) gene fusion (win6-GUS) responds to wounding locally and remotely in transgenic tobacco. These data indicate that the poplar win6 promoter has regulatory elements that are responsive to 'wound signals' in the heterologous host. In addition, win6-GUS is developmentally activated in unwounded young leaves and floral tissues of transgenic tobacco. Similar developmental expression patterns are found to occur for win6 in poplar trees, demonstrating that a herbaceous plant can serve as a host for woody tree transgene analysis and can accurately predict expression patterns in tree tissues (e.g. flowers) that would be difficult to study in free-living trees.

Adaptation, Biological↗

Anaerobic induction of the maize GapC4 promoter in poplar leaves requires light and high CO2.

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 ( GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula x P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO2 atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO2 and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO2 concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.

Aerobiosis↗

Gene expression profiling of systemically wound-induced defenses in hybrid poplar.

As part of an ongoing effort to identify genes involved in poplar defense responses, and to provide a resource for comparative analysis of woody and non-woody plant defense, we generated expressed sequence tags (ESTs) from a library constructed from systemically wounded leaves of hybrid poplar (Populus trichocarpa x P. deltoides). Partial sequences were obtained from the 5' ends of 928 individual cDNAs, which could be grouped into 565 non-overlapping sequences. Of these, 447 sequences were singletons, while the remainder fell into 118 clusters containing up to 17 partially overlapping ESTs. Approximately 81% of the EST sequences showed similarity to previously described sequences in public databases. Of these, the distribution of gene functions within the EST set indicated that approximately 11% of the ESTs encode proteins potentially involved in defense or secondary metabolism, while photosynthesis and primary metabolism accounted for 45% of the expressed genes. Two types of defense proteins, Kunitz trypsin inhibitors and chitinases, were found among the ten most abundant ESTs, indicating the significant impact of wounding on the leaf transcriptome and suggesting that these functions are important for hybrid poplar defense. In the course of this work, three new wound-inducible Kunitz trypsin inhibitor-like genes and two new chitinase-like genes were characterized. A suite of other systemically wound-induced genes were identified using northern and macroarray analysis, indicating diversity and multiplicity in the induced defense response. Overall, we demonstrate that defense-related genes of hybrid poplar have a variety of functions, and show remarkably diverse expression patterns upon wounding.

Crosses, Genetic↗

Influence of brick air scrubber by-product on growth and development of corn and hybrid poplar.

Studies were conducted to determine the effects of spent reagent from air pollution control scrubbers used at a brick manufacturing facility on emergence, growth, and physiological responses of corn and hybrid poplar plants. Scrubber by-product was obtained from General Shale Brick, Louisville, KY. Potting substrate was weighed and quantities of scrubber by-product were added to the substrate to obtain treatments of 0%, 6.25%, 12.5%, 25%, 50%, 75%, and 100% scrubber by-product (w:w) for the corn study. Each treatment mix was potted into nine replicate polyethylene pots and four corn seeds were sown per pot. The pots were randomized in a greenhouse at Clemson University and the number of seedlings emerging from each treatment, dark-adapted leaf chlorophyll a fluorescence, and shoot heights were measured at the end of a 21-day growth period. Then, dry shoot biomass was determined for plants from each treatment and plant tissues were analyzed for selected constituents. For the poplar study, nine-inch cuttings of hybrid poplar clone 15-29 (Populus trichocarpa x P. deltoides) and clone OP367 (P. deltoides x P. nigra) were planted in treatments of scrubber by-product-potting soil mixes of 0% , 5% , 10% , and 25% w:w. Leaf chlorophyll a fluorescence was measured over six weeks and cumulative leaf area, dry biomass, and nutrient content of tissues were determined upon harvest. Results of these studies indicate that percent seedling emergence for corn plants decreased with increasing scrubber by-product application rates. Application rates up to 12.5% scrubber by-product w:w had no adverse effect on corn seedling emergence. Shoot elongation, biomass production, and the status of the photosynthetic apparatus of the seedlings were also not severely impaired at applications below this level. A critical value of 58.2% w:w scrubber by-product was estimated to cause 25% inhibition of seedling emergence. Biomass production, cumulative leaf area, and chlorophyll a fluorescence of hybrid poplar plants were not affected by scrubber by-product applications of up to 5% w:w.

Biomass↗

Investigation on water consumption characteristics and water use efficiency of poplar under soil water deficits on the Loess Plateau.

The water consumption characteristics and water use efficiency (WUE) of poplar (Populus simonii) were investigated under the condition of three different soil water regimes, which were 70%, 55% and 40% of field capacity (FC), respectively. The results showed that water potential, water content, photosynthetic rate, WUE of leaves, and plant growth rate decreased significantly with reduction in soil water content. At the level of adequate and medium soil water content, rapid growth and biomass accumulation in poplar occurred during May and June, whereas this phenomenon occurred only during May under severe soil drought condition. Total water consumption and biomass growth were the greatest under adequate soil water condition, decreased under medium soil drought condition, and lowest under severe soil drought condition. Total WUE was the highest under medium soil water condition and lowest under severe soil condition. Changing patterns of water consumption for daily rate, every 10-day rate, and month rate were quite different under these three soil water regimes. For all these three treatments, the highest monthly water consumption rate occurred during July and June. The highest water consumption over a 10-day period was during in the second 10 days of July, the first 10 days of July, and the last 10 days of June for these three treatments, respectively. The day for the highest water consumption in the medium and severe drought treatments occurred 1 or 2 months earlier than the adequate soil water treatment. The daily time for the greatest water consumption was different throughout the life span of poplar under these soil water levels. According to these results, we concluded that poplar did not have the characteristics of drought-resistance plants, and we do not recommend that this tree species be planted over a wider range of the Loess Plateau in China.

Biomass↗

Metabolism and mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine inside poplar tissues (Populus deltoides x nigra DN-34).

Poplar tissue cultures and leaf crude extracts (Populus deltoides x nigra DN-34) were exposed to [U-14C]hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and incubated under light and in the dark. Poplar tissue cultures were able to partially reduce RDX to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), regardless of the presence or absence of light. However, further transformation of RDX, MNX, and DNX required exposure to light and resulted in the formation of formaldehyde (CH2O), methanol (CH3OH), and carbon dioxide (CO2). Similarly, transformation of RDX by poplar leaf crude extracts required exposure to light. Neither reduction of RDX to MNX and DNX nor mineralization into CO2 were recorded in crude extracts, even when exposed to light, suggesting that both processes were light-independent and required intact plant cells. Control experiments without plant material showed that RDX was partially transformed abiotically, by the sole action of light, but to a lesser extent than in the presence of plant crude extracts, suggesting the intervention of plant subcellular structures through a light-mediated mechanism. Poplar tissue cultures were also shown to mineralize 14CH2O and 14CH3OH, regardless of the presence or absence of light. These results suggest that transformation of [U-14C]RDX by plant tissue cultures may occur through a three-step process, involving (i) a light-independent reduction of RDX to MNX and DNX by intact plant cells; (ii) a plant/light-mediated breakdown of the heterocyclic ring of RDX, MNX, or DNX into C1-labeled metabolites (CH2O and CH3OH); and (iii) a further light-independent mineralization of C1-labeled metabolites by intact plant cells. This is the first time that a significant mineralization of RDX into CO2 by light-exposed plant tissue cultures is reported.

Biodegradation, Environmental↗