Phytochrome-interacting factors play shared and distinct roles in regulating shade avoidance responses in Populus trees.

Plants adjust their growth and development in response to changing light caused by canopy shade. The molecular mechanisms underlying shade avoidance responses have been widely studied in Arabidopsis and annual crop species, yet the shade avoidance signalling in woody perennial trees remains poorly understood. Here, we first showed that PtophyB1/2 photoreceptors serve conserved roles in attenuating the shade avoidance syndrome (SAS) in poplars. Next, we conducted a systematic identification and characterization of eight PtoPIF genes in Populus tomentosa. Knocking out different PtoPIFs led to attenuated shade responses to varying extents, whereas overexpression of PtoPIFs, particularly PtoPIF3.1 and PtoPIF3.2, led to constitutive SAS phenotypes under normal light and enhanced SAS responses under simulated shade. Notably, our results revealed that distinct from Arabidopsis PIF4 and PIF5, which are major regulators of SAS, the Populus homologues PtoPIF4.1 and PtoPIF4.2 seem to play a minor role in controlling shade responses. Moreover, we showed that PtoPIF3.1/3.2 could directly activate the expression of the auxin biosynthetic gene PtoYUC8 in response to shade, suggesting a conserved PIF-YUC-auxin pathway in modulating SAS in tree. Overall, our study provides insights into shared and divergent functions of PtoPIF members in regulating various aspects of the SAS in Populus.

Condensed tannins as antioxidants that protect poplar against oxidative stress from drought and UV-B.

Condensed tannins (CTs, proanthocyanidins) are widespread polymeric flavan-3-ols known for their ability to bind proteins. In poplar (Populus spp.), leaf condensed tannins are induced by both biotic and abiotic stresses, suggesting diverse biological functions. Here we demonstrate the ability of CTs to function as physiological antioxidants, preventing oxidative and cellular damage in response to drought and UV-B irradiation. Chlorophyll fluorescence was used to monitor photosystem II performance, and both hydrogen peroxide and malondialdehyde content was assayed as a measure of oxidative damage. Transgenic MYB-overexpressing poplar (Populus tremula × P. tremuloides) with high CT content showed reduced photosystem damage and lower hydrogen peroxide and malondialdehyde content after drought and UV-B stress. This antioxidant effect of CT was observed using two different poplar MYB CT regulators, in multiple independent lines and different genetic backgrounds. Additionally, low-CT MYB134-RNAi transgenic poplars showed enhanced susceptibility to drought-induced oxidative stress. UV-B radiation had different impacts than drought on chlorophyll fluorescence, but all high-CT poplar lines displayed reduced sensitivity to both stresses. Our data indicate that CTs are significant defences against oxidative stress. The broad distribution of CTs in forest systems that are exposed to diverse abiotic stresses suggests that these compounds have wider functional roles than previously realized.

A poplar B-box protein PtrBBX23 modulates the accumulation of anthocyanins and proanthocyanidins in response to high light.

Flavonoids, which modulate plant resistance to various stresses, can be induced by high light. B-box (BBX) transcription factors (TFs) play crucial roles in the transcriptional regulation of flavonoids biosynthesis, but limited information is available on the association of BBX proteins with high light. We present a detailed overview of 45 Populus trichocarpa BBX TFs. Phylogenetic relationships, gene structure, tissue-specific expression patterns and expression profiles were determined under 10 stress or phytohormone treatments to screen candidate BBX proteins associated with the flavonoid pathway. Sixteen candidate genes were identified, of which five were expressed predominantly in young leaves and roots, and BBX23 showed the most distinct response to high light. Overexpression of BBX23 in poplar activated expression of MYB TFs and structural genes in the flavonoid pathway, thereby promoting the accumulation of proanthocyanidins and anthocyanins. CRISPR/Cas9-generated knockout of BBX23 resulted in the opposite trend. Furthermore, the phenotype induced by BBX23 overexpression was enhanced under exposure to high light. BBX23 was capable of binding directly to the promoters of proanthocyanidin- and anthocyanin-specific genes, and its interaction with HY5 enhanced activation activity. We identified novel regulators of flavonoid biosynthesis in poplar, thereby enhancing our general understanding of the transcriptional regulatory mechanisms involved.

Identification and characterization of the CONSTANS-like gene family and its expression profiling under light treatment in Populus.

The CONSTANS-like (COL) genes play an important role in the photoperiodic flowering pathway. Poplar is a perennial woody plant with a long juvenile phase, but the molecular characterization of COL genes in Populus is limited. In this study, 14 COL genes were identified in the Populus genome. Phylogenetic analysis indicated the PtCOL proteins were divided into three subgroups, and the members of each subgroup had similar gene structure and motif composition. Chromosome distribution analysis showed that 14 PtCOL genes were distributed on 10 chromosomes. Multiple sequence alignment indicated that these proteins contained a highly conserved B-box1 and a conserved CCT domain, but the B-box2 structure was divided into three different types. Promoter analysis found that there were several light-responsive cis-elements in the PtCOL genes. Furthermore, tissue-specific expression showed that all nine PtCOL genes were widely expressed in various tissues and organs of Populus, and were preferentially expressed in the leaves. Additionally, the transcription level of PtCOL exhibited a diurnal oscillation pattern in different light conditions. This study not only provided comprehensive information for further analysis of the function of the PtCOL gene family, but also revealed the biological roles of PtCOL genes in the photoperiod-dependent flowering process of Populus.

Efficient evaluation of a gene containment system for poplar through early flowering induction.

KEY MESSAGE: The early flowering system HSP::AtFT allowed a fast evaluation of a gene containment system based on the construct PsEND1::barnase-barstar for poplar. Transgenic lines showed disturbed pollen development and sterility. Vertical gene transfer through pollen flow from transgenic or non-native plant species into their crossable natural relatives is a major concern. Gene containment approaches have been proposed to reduce or even avoid gene flow among tree species. However, evaluation of genetic containment strategies for trees is very difficult due to the long-generation times. Early flowering induction would allow faster evaluation of genetic containment in this case. Although no reliable methods were available for the induction of fertile flowers in poplar, recently, a new early flowering approach was developed. In this study, early flowering poplar lines containing the gene construct PsEND1::barnase-barstar were obtained. The PsEND1 promoter was chosen due to its early expression pattern, its versality and efficiency for generation of male-sterile plants fused to the barnase gene. RT-PCRs confirmed barnase gene activity in flowers, and pollen development was disturbed, leading to sterile flowers. The system developed in this study represents a valuable tool for gene containment studies in forest tree species.

Conjoint Analysis of Genome-Wide lncRNA and mRNA Expression of Heteromorphic Leavesin Response to Environmental Heterogeneityin Populus euphratica.

Heterophylly is the phenomenon of leaf forms varying along the longitudinal axis within a single plant. Populus euphratica, a heterophyllous woody plant, develops lanceolate leaves and dentate broad-ovate leaves on the bottom and top of the canopy, respectively, which are faced with different intensities of ambient solar radiation. However, the mechanism of the heteromorphic leaf response to the microenvironment in P. euphratica remains elusive. Here, we show that the dentate broad-ovate leaves have advantages in tolerating high light intensity, while lanceolate leaves are excellent at capturing light. Compared with lanceolate leaves, more trichomes, higher stomatal density, thicker lamina, and higher specific leaf weight were observed in dentate broad-ovate leaves. Furthermore, high-throughput RNA sequencing analysis revealed that the expression patterns of genes and long noncoding RNAs (lncRNAs) are different between the two heteromorphic leaves. A total of 36,492 genes and 1725 lncRNAs were detected, among which 586 genes and 54 lncRNAs were differentially expressed. Based on targets prediction, lncRNAs and target genes involved in light adaption, protein repair, stress response, and growth and development pathways were differentially expressed in heteromorphic leaves, 10 pairs of which were confirmed by quantitative real-time PCR. Additionally, the analysis of interactions indicated that lncRNA-mRNA interactions were involved in the response to the microenvironment of heteromorphic leaves. Taken together, these results suggest that the morphological features and joint regulation of lncRNA-mRNA in heteromorphic leaves may serve as survival strategies for P. euphratica, which could lead to optimal utilization of environmental factors.

Interannual variation in UV-B and temperature effects on bud phenology and growth in Populus tremula.

Warming affects phenological processes such as spring bud break and autumnal bud set, and also growth rates of trees. Recently, it has been shown that these physiological processes also may be influenced by the ultraviolet-B (UV-B) part of the solar spectrum, and there are reasons to expect that the two environmental factors induce interactive effects when acting in concert. In this study, our aim was to elucidate how experimental enhancements in temperature and UV-B, alone and in combination, affect growth and seasonal phenology of Eurasian aspen (Populus tremula) over several growing seasons (three years). Moreover, we tested how environmentally induced changes in phenology affect the growth achieved over each season, that is, the importance of a prolonged growing season for growth yield. The plants grew in an outdoor experiment with modulated enhancements of temperature and UV-B during the growing season. Both UV-B and temperature enhancement affected bud set dates, while bud break dates were only affected by temperature enhancement. Temperature delayed bud set in all years, but gradually less over years, while UV-B yielded earlier bud set the first year but showed a delayed response the following years. Bud break was always earlier under temperature enhancement. The experimentally induced extension of the growing season in both ends had a positive effect of growth throughout the three-year period. However, the reduced responsiveness of bud set to both enhancement treatments suggest that the plants gradually acclimated to the modified climate, a finding that should also be investigated for other tree species.

Genetic variation in transcription factors and photosynthesis light-reaction genes regulates photosynthetic traits.

Transcription factors (TFs) play crucial roles in regulating the production of the components required for photosynthesis; elucidating the mechanisms by which underlying genetic variation in TFs affects complex photosynthesis-related traits may improve our understanding of photosynthesis and identify ways to improve photosynthetic efficiency. Promoter analysis of 96 nuclear-encoded Populus tomentosa Carr. genes within this pathway revealed 47 motifs responsive to light, stress, hormones and organ-specific regulation, as well as 86 TFs that might bind these motifs. Using phenotype-genotype associations, we identified 244 single-nucleotide polymorphisms (SNPs) within 105 genes associated with 12 photosynthesis-related traits. Most (30.33%) of these SNPs were located in intronic regions and these SNPs explained 18.66% of the mean phenotypic variation in the photosynthesis-related traits. Additionally, expression quantitative trait loci (eQTL) mapping identified 216 eQTLs associated with 110 eGenes (genes regulated by eQTLs), explaining 14.12% of the variability of gene expression. The lead SNPs of 12.04% of the eQTLs also contributed to phenotypic variation. Among these, a SNP in zf-Dof 5.6 (G120_9287) affected photosynthesis by modulating the expression of a sub-regulatory network of eight other TFs, which in turn regulate 55 photosynthesis-related genes. Furthermore, epistasis analysis identified a large interacting network representing 732 SNP-SNP pairs, of which 354 were photosynthesis gene-TF pairs, emphasizing the important roles of TFs in affecting photosynthesis-related traits. We combined eQTL and epistasis analysis and found 32 TFs harboring eQTLs being epistatic to their targets (identified by eQTL analysis), of which 15 TFs were also associated with photosynthesis traits. We therefore constructed a schematic model of TFs involved in regulating the photosynthetic light reaction pathway. Taken together, our results provide insight into the genetic regulation of photosynthesis, and may drive progress in the marker-assisted selection of desirable P. tomentosa genotypes with more efficient photosynthesis.

More than just CO2 -recycling: corticular photosynthesis as a mechanism to reduce the risk of an energy crisis induced by low oxygen.

Reassimilation of internal CO2 via corticular photosynthesis (PScort ) has an important effect on the carbon economy of trees. However, little is known about its role as a source of O2 supply to the stem parenchyma and its implications in consumption and movement of O2 within trees. PScort of young Populus nigra (black poplar) trees was investigated by combining optical micro-optode measurements with monitoring of stem chlorophyll fluorescence. During times of zero sap flow in spring, stem oxygen concentrations (cO2 ) exhibited large temporal changes. In the sapwood, over 80% of diurnal changes in cO2 could be explained by respiration rates (Rd(mod) ). In the cortex, photosynthetic oxygen release during the day altered this relationship. With daytime illumination, oxygen levels in the cortex steadily increased from subambient and even exhibited a diel period of superoxia of up to 110% (% air sat.). By contrast, in the sapwood, cO2 never reached ambient levels; the diurnal oxygen deficit was up to 25% of air saturation. Our results confirm that PScort is not only a CO2 -recycling mechanism, it is also a mechanism to actively raise the cortical O2 concentration and counteract temporal/spatial hypoxia inside plant stems.

Dying piece by piece: carbohydrate dynamics in aspen (Populus tremuloides) seedlings under severe carbon stress.

Carbon starvation as a mechanism of tree mortality is poorly understood. We exposed seedlings of aspen (Populus tremuloides) to complete darkness at 20 or 28 °C to identify minimum non-structural carbohydrate (NSC) concentrations at which trees die and to see if these levels vary between organs or with environmental conditions. We also first grew seedlings under different shade levels to determine if size affects survival time under darkness due to changes in initial NSC concentration and pool size and/or respiration rates. Darkness treatments caused a gradual dieback of tissues. Even after half the stem had died, substantial starch reserves were still present in the roots (1.3-3% dry weight), indicating limitations to carbohydrate remobilization and/or transport during starvation in the absence of water stress. Survival time decreased with increased temperature and with increasing initial shade level, which was associated with smaller biomass, higher respiration rates, and initially smaller NSC pool size. Dead tissues generally contained no starch, but sugar concentrations were substantially above zero and differed between organs (~2% in stems up to ~7.5% in leaves) and, at times, between temperature treatments and initial, pre-darkness shade treatments. Minimum root NSC concentrations were difficult to determine because dead roots quickly began to decompose, but we identify 5-6% sugar as a potential threshold for living roots. This variability may complicate efforts to identify critical NSC thresholds below which trees starve.

Comparative leaf growth strategies in response to low-water and low-light availability: variation in leaf physiology underlies variation in leaf mass per area in Populus tremuloides.

Developmental phenotypic plasticity can allow plants to buffer the effects of abiotic and biotic environmental stressors. Therefore, it is vital to improve our understanding of how phenotypic plasticity in ecological functional traits is coordinated with variation in physiological performance in plants. To identify coordinated leaf responses to low-water (LW) versus low-light (LL) availability, we measured leaf mass per area (LMA), leaf anatomical characteristics and leaf gas exchange of juvenile Populus tremuloides Michx. trees. Spongy mesophyll tissue surface area (Asmes/A) was correlated with intrinsic water-use efficiency (WUEi: photosynthesis, (Aarea)/stomatal conductance (gs)). Under LW availability, these changes occurred at the cost of greater leaf tissue density and reduced expansive growth, as leaves were denser but were only 20% the final area of control leaves, resulting in elevated LMA and elevated WUEi. Low light resulted in reduced palisade mesophyll surface area (Apmes/A) while spongy mesophyll surface area was maintained (Asmes/A), with no changes to WUEi. These leaf morphological changes may be a plastic strategy to increase laminar light capture while maintaining WUEi. With reduced density and thickness, however, leaves were 50% the area of control leaves, ultimately resulting in reduced LMA. Our results illustrate that P. tremuloides saplings partially maintain physiological function in response to water and light limitation by inducing developmental plasticity in LMA with underlying anatomical changes. We discuss additional implications of these results in the context of developmental plasticity, growth trade-offs and the ecological impacts of climate change.

Blue light differentially represses mesophyll conductance in high vs low latitude genotypes of Populus trichocarpa Torr. & Gray.

To explore what role chloroplast positioning might have in relation to latitudinal variation in mesophyll conductance (gm) of Populus trichocarpa Torr. & Gray (black cottonwood), we examined photosynthetic response to different blue light treatments in six representative genotypes (three northern and three southern). The proportion of blue (B) to red light was varied from 0:100, 10:90, 20:80, 40:60, and 60:40 while keeping the total photosynthetic photon flux density constant. Mesophyll conductance was estimated by monitoring chlorophyll fluorescence in combination with gas exchange. Compared to the control (10% B), gm was significantly lower with increasing blue light. Consistent with a change in chloroplast positioning, there was a simultaneous but reversible decrease in chlorophyll content index (CCI), as measured by foliar greenness, while the extracted, actual chlorophyll content (ACC) remained unchanged. Blue-light-induced decreases in gm and CCI were greater in northern genotypes than in southern genotypes, both absolutely and proportionally, consistent with their inherently higher photosynthetic rate. Treatment of leaves with cytochalasin D, an inhibitor of actin-based chloroplast motility, reduced both CCI and ACC but had no effect on the CCI/ACC ratio and fully blocked any effect of blue light on CCI. Cytochalasin D reduced gm by ∼56% under 10% B, but did not block the effect of 60% B on gm, which was reduced a further 20%. These results suggest that the effect of high blue light on gm is at least partially independent of chloroplast repositioning. High blue light reduced carbonic anhydrase activity by 20% (P<0.05), consistent with a possible reduction in protein-mediated facilitation of CO2 diffusion.

Comparative transcriptomic analysis reveals the roles of overlapping heat-/drought-responsive genes in poplars exposed to high temperature and drought.

High temperature (HT) and drought are both critical factors that constrain tree growth and survival under global climate change, but it is surprising that the transcriptomic reprogramming and physiological relays involved in the response to HT and/or drought remain unknown in woody plants. Thus, Populus simonii saplings were exposed to either ambient temperature or HT combined with sufficient watering or drought. RNA-sequencing analysis showed that a large number of genes were differentially expressed in poplar roots and leaves in response to HT and/or desiccation, but only a small number of these genes were identified as overlapping heat-/drought-responsive genes that are mainly involved in RNA regulation, transport, hormone metabolism, and stress. Furthermore, the overlapping heat-/drought-responsive genes were co-expressed and formed hierarchical genetic regulatory networks under each condition compared. HT-/drought-induced transcriptomic reprogramming is linked to physiological relays in poplar roots and leaves. For instance, HT- and/or drought-induced abscisic acid accumulation and decreases in auxin and other phytohormones corresponded well with the differential expression of a few genes involved in hormone metabolism. These results suggest that overlapping heat-/drought-responsive genes will play key roles in the transcriptional and physiological reconfiguration of poplars to HT and/or drought under future climatic scenarios.

Short day length-induced decrease of cesium uptake without altering potassium uptake manner in poplar.

Short day length-induced alteration of potassium (K) localization in perennial trees is believed to be a mechanism for surviving and adapting to severe winters. To investigate the relationship between cesium (Cs) and K localizations, a model tree poplar, hybrid aspen T89, was employed. Under short day length conditions, the amount of 137Cs absorbed through the root and translocated to the root was drastically reduced, but 42K was not. Potassium uptake from the rhizosphere is mediated mainly by KUP/HAK/KT and CNGC transporters. In poplar, however, these genes were constantly expressed under short-day conditions except for a slight increase in the expression a KUP/HAK/KT gene six weeks after the onset of the short-day treatment. These results indicated that the suppression of 137Cs uptake was triggered by short day length but not regulated by competitive Cs+ and K+ transport. We hypothesize that there are separately regulated Cs+ and K+ transport systems in poplar.

Moderate salt treatment alleviates ultraviolet-B radiation caused impairment in poplar plants.

The effects of moderate salinity on the responses of woody plants to UV-B radiation were investigated using two Populus species (Populus alba and Populus russkii). Under UV-B radiation, moderate salinity reduced the oxidation pressure in both species, as indicated by lower levels of cellular H2O2 and membrane peroxidation, and weakened the inhibition of photochemical efficiency expressed by O-J-I-P changes. UV-B-induced DNA lesions in chloroplast and nucleus were alleviated by salinity, which could be explained by the higher expression levels of DNA repair system genes under UV-B&salt condition, such as the PHR, DDB2, and MutSα genes. The salt-induced increase in organic osmolytes proline and glycine betaine, afforded more efficient protection against UV-B radiation. Therefore moderate salinity induced cross-tolerance to UV-B stress in poplar plants. It is thus suggested that woody plants growing in moderate salted condition would be less affected by enhanced UV-B radiation than plants growing in the absence of salt. Our results also showed that UV-B signal genes in poplar plants PaCOP1, PaSTO and PaSTH2 were quickly responding to UV-B radiation, but not to salt. The transcripts of PaHY5 and its downstream pathway genes (PaCHS1, PaCHS4, PaFLS1 and PaFLS2) were differently up-regulated by these treatments, but the flavonoid compounds were not involved in the cross-tolerance since their concentration increased to the same extent in both UV-B and combined stresses.

The effect of warming and enhanced ultraviolet radiation on gender-specific emissions of volatile organic compounds from European aspen.

Different environmental stress factors often occur together but their combined effects on plant secondary metabolism are seldom considered. We studied the effect of enhanced ultraviolet (UV-B) (31% increase) radiation and temperature (ambient +2 °C) singly and in combination on gender-specific emissions of volatile organic compounds (VOCs) from 2-year-old clones of European aspen (Populus tremula L.). Plants grew in 36 experimental plots (6 replicates for Control, UV-A, UV-B, T, UV-A+T and UV-B+T treatments), in an experimental field. VOCs emitted from shoots were sampled from two (1 male and 1 female) randomly selected saplings (total of 72 saplings), per plot on two sampling occasions (June and July) in 2014. There was a significant UV-B×temperature interaction effect on emission rates of different VOCs. Isoprene emission rate was increased due to warming, but warming also modified VOC responses to both UV-A and UV-B radiation. Thus, UV-A increased isoprene emissions without warming, whereas UV-B increased emissions only in combination with warming. Warming-modified UV-A and UV-B responses were also seen in monoterpenes (MTs), sesquiterpenes (SQTs) and green leaf volatiles (GLVs). MTs showed also a UV × gender interaction effect as females had higher emission rates under UV-A and UV-B than males. UV × gender and T × gender interactions caused significant differences in VOC blend as there was more variation (more GLVs and trans-ß-caryophyllene) in VOCs from female saplings compared to male saplings. VOCs from the rhizosphere were also collected from each plot in two exposure seasons, but no significant treatment effects were observed. Our results suggest that simultaneous warming and elevated-UV-radiation increase the emission of VOCs from aspen. Thus the contribution of combined environmental factors on VOC emissions may have a greater impact to the photochemical reactions in the atmosphere compared to the impact of individual factors acting alone.

Effects of heat and drought stress on post-illumination bursts of volatile organic compounds in isoprene-emitting and non-emitting poplar.

Over the last decades, post-illumination bursts (PIBs) of isoprene, acetaldehyde and green leaf volatiles (GLVs) following rapid light-to-dark transitions have been reported for a variety of different plant species. However, the mechanisms triggering their release still remain unclear. Here we measured PIBs of isoprene-emitting (IE) and isoprene non-emitting (NE) grey poplar plants grown under different climate scenarios (ambient control and three scenarios with elevated CO2 concentrations: elevated control, periodic heat and temperature stress, chronic heat and temperature stress, followed by recovery periods). PIBs of isoprene were unaffected by elevated CO2 and heat and drought stress in IE, while they were absent in NE plants. On the other hand, PIBs of acetaldehyde and also GLVs were strongly reduced in stress-affected plants of all genotypes. After recovery from stress, distinct differences in PIB emissions in both genotypes confirmed different precursor pools for acetaldehyde and GLV emissions. Changes in PIBs of GLVs, almost absent in stressed plants and enhanced after recovery, could be mainly attributed to changes in lipoxygenase activity. Our results indicate that acetaldehyde PIBs, which recovered only partly, derive from a new mechanism in which acetaldehyde is produced from methylerythritol phosphate pathway intermediates, driven by deoxyxylulose phosphate synthase activity.

Molecular Cloning and Functional Analysis of UV RESISTANCE LOCUS 8 (PeUVR8) from Populus euphratica.

Ultraviolet-B (UV-B; 280-315 nm) light, which is an integral part of the solar radiation reaching the surface of the Earth, induces a broad range of physiological responses in plants. The UV RESISTANCE LOCUS 8 (UVR8) protein is the first and only light photoreceptor characterized to date that is specific for UV-B light and it regulates various aspects of plant growth and development in response to UV-B light. Despite its involvement in the control of important plant traits, most studies on UV-B photoreceptors have focused on Arabidopsis and no data on UVR8 function are available for forest trees. In this study, we isolated a homologue of the UV receptor UVR8 of Arabidopsis, PeUVR8, from Populus euphratica (Euphrates poplar) and analyzed its structure and function in detail. The deduced PeUVR8 amino acid sequence contained nine well-conserved regulator of chromosome condensation 1 (RCC1) repeats and the region 27 amino acids from the C terminus (C27) that interact with COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC1). Secondary and tertiary structure analysis showed that PeUVR8 shares high similarity with the AtUVR8 protein from Arabidopsis thaliana. Using heterologous expression in Arabidopsis, we showed that PeUVR8 overexpression rescued the uvr8 mutant phenotype. In addition, PeUVR8 overexpression in wild-type background seedlings grown under UV-B light inhibited hypocotyl elongation and enhanced anthocyanin accumulation. Furthermore, we examined the interaction between PeUVR8 and AtCOP1 using a bimolecular fluorescence complementation (BiFC) assay. Our data provide evidence that PeUVR8 plays important roles in the control of photomorphogenesis in planta.

Controls of the quantum yield and saturation light of isoprene emission in different-aged aspen leaves.

Leaf age alters the balance between the use of end-product of plastidic isoprenoid synthesis pathway, dimethylallyl diphosphate (DMADP), in prenyltransferase reactions leading to synthesis of pigments of photosynthetic machinery and in isoprene synthesis, but the implications of such changes on environmental responses of isoprene emission have not been studied. Because under light-limited conditions, isoprene emission rate is controlled by DMADP pool size (SDMADP ), shifts in the share of different processes are expected to particularly strongly alter the light dependency of isoprene emission. We examined light responses of isoprene emission in young fully expanded, mature and old non-senescent leaves of hybrid aspen (Populus tremula x P. tremuloides) and estimated in vivo SDMADP and isoprene synthase activity from post-illumination isoprene release. Isoprene emission capacity was 1.5-fold larger in mature than in young and old leaves. The initial quantum yield of isoprene emission (αI ) increased by 2.5-fold with increasing leaf age primarily as the result of increasing SDMADP . The saturating light intensity (QI90 ) decreased by 2.3-fold with increasing leaf age, and this mainly reflected limited light-dependent increase of SDMADP possibly due to feedback inhibition by DMADP. These major age-dependent changes in the shape of the light response need consideration in modelling canopy isoprene emission.

Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen.

Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.