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Growth is characterized by the interplay between cell division and cell expansion, two processes that occur separated along the growth zone at the maize leaf. To gain further insight into the transition between cell division and cell expansion, conditions were investigated in which the position of this transition zone was positively or negatively affected. High levels of gibberellic acid (GA) in plants overexpressing the GA biosynthesis gene GA20-OXIDASE (GA20OX-1 OE ) shifted the transition zone more distally, whereas mild drought, which is associated with lowered GA biosynthesis, resulted in a more basal positioning. However, the increased levels of GA in the GA20OX-1 OE line were insufficient to convey tolerance to the mild drought treatment, indicating that another mechanism in addition to lowered GA levels is restricting growth during drought. Transcriptome analysis with high spatial resolution indicated that mild drought specifically induces a reprogramming of transcriptional regulation in the division zone. 'Leaf Growth Viewer' was developed as an online searchable tool containing the high-resolution data., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Plants have established different mechanisms to cope with environmental fluctuations and accordingly fine-tune their growth and development through the regulation of complex molecular networks. It is largely unknown how the network architectures change and what the key regulators in stress responses and plant growth are. Here, we investigated a complex, highly interconnected network of 20 Arabidopsis transcription factors (TFs) at the basis of leaf growth inhibition upon mild osmotic stress. We tracked the dynamic behavior of the stress-responsive TFs over time, showing the rapid induction following stress treatment, specifically in growing leaves. The connections between the TFs were uncovered using inducible overexpression lines and were validated with transient expression assays. This study resulted in the identification of a core network, composed of ERF6, ERF8, ERF9, ERF59, and ERF98, which is responsible for most transcriptional connections. The analyses highlight the biological function of this core network in environmental adaptation and its redundancy. Finally, a phenotypic analysis of loss-of-function and gain-of-function lines of the transcription factors established multiple connections between the stress-responsive network and leaf growth., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Maize is the highest yielding cereal crop grown worldwide for grain or silage. Here, we show that modulating the expression of the maize PLASTOCHRON1 (ZmPLA1) gene, encoding a cytochrome P450 (CYP78A1), results in increased organ growth, seedling vigour, stover biomass and seed yield. The engineered trait is robust as it improves yield in an inbred as well as in a panel of hybrids, at several locations and over multiple seasons in the field. Transcriptome studies, hormone measurements and the expression of the auxin responsive DR5rev:mRFPer marker suggest that PLA1 may function through an increase in auxin. Detailed analysis of growth over time demonstrates that PLA1 stimulates the duration of leaf elongation by maintaining dividing cells in a proliferative, undifferentiated state for a longer period of time. The prolonged duration of growth also compensates for growth rate reduction caused by abiotic stresses.

Plants sense and integrate a variety of signals from the environment through different interacting signal transduction pathways that involve hormones and signaling molecules. Using ALTERNATIVE OXIDASE1a ( AOX1a ) gene expression as a model system of retrograde or stress signaling between mitochondria and the nucleus, MYB DOMAIN PROTEIN29 (MYB29) was identified as a negative regulator ( regulator of alternative oxidase1a 7 [ rao7 ] mutant) in a genetic screen of Arabidopsis ( Arabidopsis thaliana ). rao7/myb29 mutants have increased levels of AOX1a transcript and protein compared to wild type after induction with antimycin A. A variety of genes previously associated with the mitochondrial stress response also display enhanced transcript abundance, indicating that RAO7/MYB29 negatively regulates mitochondrial stress responses in general. Meta-analysis of hormone-responsive marker genes and identification of downstream transcription factor networks revealed that MYB29 functions in the complex interplay of ethylene, jasmonic acid, salicylic acid, and reactive oxygen species signaling by regulating the expression of various ETHYLENE RESPONSE FACTOR and WRKY transcription factors. Despite an enhanced induction of mitochondrial stress response genes, rao7 / myb29 mutants displayed an increased sensitivity to combined moderate light and drought stress. These results uncover interactions between mitochondrial retrograde signaling and the regulation of glucosinolate biosynthesis, both regulated by RAO7/MYB29. This common regulator can explain why perturbation of the mitochondrial function leads to transcriptomic responses overlapping with responses to biotic stress., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The final size of plant organs is determined by a combination of cell proliferation and cell expansion. Leaves account for a large part of above-ground biomass and provide energy to complete the plant's life cycle. Although the final size of leaves is remarkably constant under fixed environmental conditions, several genes have been described to enhance leaf growth when their expression is modulated. In Arabidopsis (Arabidopsis thaliana), mutations in DA1 and BB increase leaf size, an effect that is synergistically enhanced in the double mutant. Here, we show that overexpression of a dominant-negative version of DA1 enhances leaf size in a broad range of natural accessions of this species, indicating a highly conserved role of this protein in controlling organ size. We also found that during early stages of development, leaves of da1-1 and bb/eod1-2 mutants were already larger than the isogenic Col-0 wild type, but this phenotype was triggered by different cellular mechanisms. Later during development, da1-1 and bb/eod1-2 leaves showed a prolonged longevity, which was enhanced in the double mutant. Conversely, ectopic expression of DA1 or BB restricted growth and promoted leaf senescence. In concert, shortly upon induction of DA1 and BB expression, several marker genes for the transition from proliferation to expansion were highly up-regulated. Additionally, multiple genes involved in maintaining the mitotic cell cycle were rapidly down-regulated and senescence genes were strongly up-regulated, particularly upon BB induction. With these results, we demonstrate that DA1 and BB restrict leaf size and promote senescence through converging and different mechanisms., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Although phytohormones such as gibberellins are essential for many conserved aspects of plant physiology and development, plants vary greatly in their responses to these regulatory compounds. Here, we use genetic perturbation of endogenous gibberellin levels to probe the extent of intraspecific variation in gibberellin responses in natural accessions of Arabidopsis (Arabidopsis thaliana). We find that these accessions vary greatly in their ability to buffer the effects of overexpression of GA20ox1, encoding a rate-limiting enzyme for gibberellin biosynthesis, with substantial differences in bioactive gibberellin concentrations as well as transcriptomes and growth trajectories. These findings demonstrate a surprising level of flexibility in the wiring of regulatory networks underlying hormone metabolism and signaling., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

We developed a bio-inspired robot controller combining an artificial genome with an agent-based control system. The genome encodes a gene regulatory network (GRN) that is switched on by environmental cues and, following the rules of transcriptional regulation, provides output signals to actuators. Whereas the genome represents the full encoding of the transcriptional network, the agent-based system mimics the active regulatory network and signal transduction system also present in naturally occurring biological systems. Using such a design that separates the static from the conditionally active part of the gene regulatory network contributes to a better general adaptive behaviour. Here, we have explored the potential of our platform with respect to the evolution of adaptive behaviour, such as preying when food becomes scarce, in a complex and changing environment and show through simulations of swarm robots in an A-life environment that evolution of collective behaviour likely can be attributed to bio-inspired evolutionary processes acting at different levels, from the gene and the genome to the individual robot and robot population., Competing Interests: Yves Van de Peer and Kathleen Marchal are Academic Editors for PeerJ.

In the last decade, microarray studies have delivered extensive inventories of transcriptome-wide changes in messenger RNA levels provoked by various types of oxidative stress in Arabidopsis (Arabidopsis thaliana). Previous cross-study comparisons indicated how different types of reactive oxygen species (ROS) and their subcellular accumulation sites are able to reshape the transcriptome in specific manners. However, these analyses often employed simplistic statistical frameworks that are not compatible with large-scale analyses. Here, we reanalyzed a total of 79 Affymetrix ATH1 microarray studies of redox homeostasis perturbation experiments. To create hierarchy in such a high number of transcriptomic data sets, all transcriptional profiles were clustered on the overlap extent of their differentially expressed transcripts. Subsequently, meta-analysis determined a single magnitude of differential expression across studies and identified common transcriptional footprints per cluster. The resulting transcriptional footprints revealed the regulation of various metabolic pathways and gene families. The RESPIRATORY BURST OXIDASE HOMOLOG F-mediated respiratory burst had a major impact and was a converging point among several studies. Conversely, the timing of the oxidative stress response was a determining factor in shaping different transcriptome footprints. Our study emphasizes the need to interpret transcriptomic data sets in a systematic context, where initial, specific stress triggers can converge to common, aspecific transcriptional changes. We believe that these refined transcriptional footprints provide a valuable resource for assessing the involvement of ROS in biological processes in plants., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Leaves are the plant's powerhouses, providing energy for all organs through sugar production during photosynthesis. However, sugars serve not only as a metabolic energy source for sink tissues but also as signaling molecules, affecting gene expression through conserved signaling pathways to regulate plant growth and development. Here, we describe an in vitro experimental assay, allowing one to alter the sucrose (Suc) availability during early Arabidopsis (Arabidopsis thaliana) leaf development, with the aim to identify the affected cellular and molecular processes. The transfer of seedlings to Suc-containing medium showed a profound effect on leaf growth by stimulating cell proliferation and postponing the transition to cell expansion. Furthermore, rapidly after transfer to Suc, mesophyll cells contained fewer and smaller plastids, which are irregular in shape and contain fewer starch granules compared with control mesophyll cells. Short-term transcriptional responses after transfer to Suc revealed the repression of well-known sugar-responsive genes and multiple genes encoded by the plastid, on the one hand, and up-regulation of a GLUCOSE-6-PHOSPHATE TRANSPORTER (GPT2), on the other hand. Mutant gpt2 seedlings showed no stimulation of cell proliferation and no repression of chloroplast-encoded transcripts when transferred to Suc, suggesting that GPT2 plays a critical role in the Suc-mediated effects on early leaf growth. Our findings, therefore, suggest that induction of GPT2 expression by Suc increases the import of glucose-6-phosphate into the plastids that would repress chloroplast-encoded transcripts, restricting chloroplast differentiation. Retrograde signaling from the plastids would then delay the transition to cell expansion and stimulate cell proliferation., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Although the response of plants exposed to severe drought stress has been studied extensively, little is known about how plants adapt their growth under mild drought stress conditions. Here, we analyzed the leaf and rosette growth response of six Arabidopsis (Arabidopsis thaliana) accessions originating from different geographic regions when exposed to mild drought stress. The automated phenotyping platform WIWAM was used to impose stress early during leaf development, when the third leaf emerges from the shoot apical meristem. Analysis of growth-related phenotypes showed differences in leaf development between the accessions. In all six accessions, mild drought stress reduced both leaf pavement cell area and number without affecting the stomatal index. Genome-wide transcriptome analysis (using RNA sequencing) of early developing leaf tissue identified 354 genes differentially expressed under mild drought stress in the six accessions. Our results indicate the existence of a robust response over different genetic backgrounds to mild drought stress in developing leaves. The processes involved in the overall mild drought stress response comprised abscisic acid signaling, proline metabolism, and cell wall adjustments. In addition to these known severe drought-related responses, 87 genes were found to be specific for the response of young developing leaves to mild drought stress., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Although quantitative characterization of growth phenotypes is of key importance for the understanding of essential networks driving plant growth, the majority of growth-related genes are still being identified based on qualitative visual observations and/or single-endpoint quantitative measurements. We developed an in vitro growth imaging system (IGIS) to perform time-resolved analysis of rosette growth. In this system, Arabidopsis plants are grown in Petri dishes mounted on a rotating disk, and images of each plate are taken on an hourly basis. Automated image analysis was developed in order to obtain several growth-related parameters, such as projected rosette area, rosette relative growth rate, compactness and stockiness, over time. To illustrate the use of the platform and the resulting data, we present the results for the growth response of Col-0 plants subjected to three mild stress conditions. Although the reduction in rosette area was relatively similar at 19 days after stratification, the time-lapse analysis demonstrated that plants react differently to salt, osmotic and oxidative stress. The rosette area was altered at various time points during development, and leaf movement and shape parameters were also affected differently. We also used the IGIS to analyze in detail the growth behavior of mutants with enhanced leaf size. Analysis of several growth-related parameters over time in these mutants revealed several specificities in growth behavior, underlining the high complexity of leaf growth coordination. These results demonstrate that time-resolved imaging of in vitro rosette growth generates a better understanding of growth phenotypes than endpoint measurements., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

The establishment of the photosynthetic apparatus during chloroplast development creates a high demand for iron as a redox metal. However, iron in too high quantities becomes toxic to the plant, thus plants have evolved a complex network of iron uptake and regulation mechanisms. Here, we examined whether four of the subgroup Ib basic helix-loop-helix transcription factors (bHLH38, bHLH39, bHLH100, bHLH101), previously implicated in iron homeostasis in roots, also play a role in regulating iron metabolism in developing leaves. These transcription factor genes were strongly up-regulated during the transition from cell proliferation to expansion, and thus sink-source transition, in young developing leaves of Arabidopsis thaliana. The four subgroup Ib bHLH genes also showed reduced expression levels in developing leaves of plants treated with norflurazon, indicating their expression was tightly linked to the onset of photosynthetic activity in young leaves. In addition, we provide evidence for a mechanism whereby the transcriptional regulators SAC51 and TCP20 antagonistically regulate the expression of these four subgroup Ib bHLH genes. A loss-of-function mutant analysis also revealed that single mutants of bHLH38, bHLH39, bHLH100, and bHLH101 developed smaller rosettes than wild-type plants in soil. When grown in agar plates with reduced iron concentration, triple bhlh39 bhlh100 bhlh101 mutant plants were smaller than wild-type plants. However, measurements of the iron content in single and multiple subgroup Ib bHLH genes, as well as transcript profiling of iron response genes during early leaf development, do not support a role for bHLH38, bHLH39, bHLH100, and bHLH101 in iron homeostasis during early leaf development.

Several genes positively influence final leaf size in Arabidopsis when mutated or overexpressed. The connections between these growth regulators are still poorly understood although such knowledge would further contribute to understand the processes driving leaf growth. In this study, we performed a combinatorial screen with 13 transgenic Arabidopsis lines with an increased leaf size. We found that from 61 analyzed combinations, 39% showed an additional increase in leaf size and most resulted from a positive epistasis on growth. Similar to what is found in other organisms in which such an epistasis assay was performed, only few genes were highly connected in synergistic combinations as we observed a positive epistasis in the majority of the combinations with samba, BRI1(OE) or SAUR19(OE). Furthermore, positive epistasis was found with combinations of genes with a similar mode of action, but also with genes which affect distinct processes, such as cell proliferation and cell expansion.DOI: http://dx.doi.org/10.7554/eLife.02252.001., (Copyright © 2014, Vanhaeren et al.)

Lignin is one of the main factors determining recalcitrance to enzymatic processing of lignocellulosic biomass. Poplars (Populus tremula x Populus alba) down-regulated for cinnamoyl-CoA reductase (CCR), the enzyme catalyzing the first step in the monolignol-specific branch of the lignin biosynthetic pathway, were grown in field trials in Belgium and France under short-rotation coppice culture. Wood samples were classified according to the intensity of the red xylem coloration typically associated with CCR down-regulation. Saccharification assays under different pretreatment conditions (none, two alkaline, and one acid pretreatment) and simultaneous saccharification and fermentation assays showed that wood from the most affected transgenic trees had up to 161% increased ethanol yield. Fermentations of combined material from the complete set of 20-mo-old CCR-down-regulated trees, including bark and less efficiently down-regulated trees, still yielded ∼ 20% more ethanol on a weight basis. However, strong down-regulation of CCR also affected biomass yield. We conclude that CCR down-regulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome.

Background: Interest in the detailed lignin and polysaccharide composition of plant cell walls has surged within the past decade partly as a result of biotechnology research aimed at converting biomass to biofuels. High-resolution, solution-state 2D 1H-13C HSQC NMR spectroscopy has proven to be an effective tool for rapid and reproducible fingerprinting of the numerous polysaccharides and lignin components in unfractionated plant cell wall materials, and is therefore a powerful tool for cell wall profiling based on our ability to simultaneously identify and comparatively quantify numerous components within spectra generated in a relatively short time. However, assigning peaks in new spectra, integrating them to provide relative component distributions, and producing color-assigned spectra, are all current bottlenecks to the routine use of such NMR profiling methods., Results: We have assembled a high-throughput software platform for plant cell wall profiling that uses spectral deconvolution by Fast Maximum Likelihood Reconstruction (FMLR) to construct a mathematical model of the signals present in a set of related NMR spectra. Combined with a simple region of interest (ROI) table that maps spectral regions to NMR chemical shift assignments of chemical entities, the reconstructions can provide rapid and reproducible fingerprinting of numerous polysaccharide and lignin components in unfractionated cell wall material, including derivation of lignin monomer unit (S:G:H) ratios or the so-called SGH profile. Evidence is presented that ROI-based amplitudes derived from FMLR provide a robust feature set for subsequent multivariate analysis. The utility of this approach is demonstrated on a large transgenic study of Arabidopsis requiring concerted analysis of 91 ROIs (including both assigned and unassigned regions) in the lignin and polysaccharide regions of almost 100 related 2D 1H-13C HSQC spectra., Conclusions: We show that when a suitable number of replicates are obtained per sample group, the correlated patterns of enriched and depleted cell wall components can be reliably and objectively detected even prior to multivariate analysis. The analysis methodology has been implemented in a publicly-available, cross-platform (Windows/Mac/Linux), web-enabled software application that enables researchers to view and publish detailed annotated spectra in addition to summary reports in simple spreadsheet data formats. The analysis methodology is not limited to studies of plant cell walls but is amenable to any NMR study where ROI segmentation techniques generate meaningful results.Please see Research Article: http://www.biotechnologyforbiofuels.com/content/6/1/46/.

Background: Second-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield. Saccharification models were built to elucidate which cell wall parameters played a role in cell wall recalcitrance., Results: Although lignin is a key polymer providing the strength necessary for the plant's ability to grow upward, a reduction in lignin content down to 64% of the wild-type level in Arabidopsis was tolerated without any obvious growth penalty. In contrast to common perception, we found that a reduction in lignin was not compensated for by an increase in cellulose, but rather by an increase in matrix polysaccharides. In most lignin mutants, the saccharification yield was improved by up to 88% cellulose conversion for the cinnamoyl-coenzyme A reductase1 mutants under pretreatment conditions, whereas the wild-type cellulose conversion only reached 18%. The saccharification models and Pearson correlation matrix revealed that the lignin content was the main factor determining the saccharification yield. However, also lignin composition, matrix polysaccharide content and composition, and, especially, the xylose, galactose, and arabinose contents influenced the saccharification yield. Strikingly, cellulose content did not significantly affect saccharification yield., Conclusions: Although the lignin content had the main effect on saccharification, also other cell wall factors could be engineered to potentially increase the cell wall processability, such as the galactose content. Our results contribute to a better understanding of the effect of lignin perturbations on plant cell wall composition and its influence on saccharification yield, and provide new potential targets for genetic improvement.

Lignin engineering is an attractive strategy to improve lignocellulosic biomass quality for processing to biofuels and other bio-based products. However, lignin engineering also results in profound metabolic consequences in the plant. We used a systems biology approach to study the plant's response to lignin perturbations. To this end, inflorescence stems of 20 Arabidopsis thaliana mutants, each mutated in a single gene of the lignin biosynthetic pathway (phenylalanine ammonia-lyase1 [PAL1], PAL2, cinnamate 4-hydroxylase [C4H], 4-coumarate:CoA ligase1 [4CL1], 4CL2, caffeoyl-CoA O-methyltransferase1 [CCoAOMT1], cinnamoyl-CoA reductase1 [CCR1], ferulate 5-hydroxylase [F5H1], caffeic acid O-methyltransferase [COMT], and cinnamyl alcohol dehydrogenase6 [CAD6], two mutant alleles each), were analyzed by transcriptomics and metabolomics. A total of 566 compounds were detected, of which 187 could be tentatively identified based on mass spectrometry fragmentation and many were new for Arabidopsis. Up to 675 genes were differentially expressed in mutants that did not have any obvious visible phenotypes. Comparing the responses of all mutants indicated that c4h, 4cl1, ccoaomt1, and ccr1, mutants that produced less lignin, upregulated the shikimate, methyl-donor, and phenylpropanoid pathways (i.e., the pathways supplying the monolignols). By contrast, f5h1 and comt, mutants that provoked lignin compositional shifts, downregulated the very same pathways. Reductions in the flux to lignin were associated with the accumulation of various classes of 4-O- and 9-O-hexosylated phenylpropanoids. By combining metabolomic and transcriptomic data in a correlation network, system-wide consequences of the perturbations were revealed and genes with a putative role in phenolic metabolism were identified. Together, our data provide insight into lignin biosynthesis and the metabolic network it is embedded in and provide a systems view of the plant's response to pathway perturbations.

Growth and development are coordinated by an array of intercellular communications. Known plant signaling molecules include phytohormones and hormone peptides. Although both classes can be implicated in the same developmental processes, little is known about the interplay between phytohormone action and peptide signaling within the cellular microenvironment. We show that genes coding for small secretory peptides, designated GOLVEN (GLV), modulate the distribution of the phytohormone auxin. The deregulation of the GLV function impairs the formation of auxin gradients and alters the reorientation of shoots and roots after a gravity stimulus. Specifically, the GLV signal modulates the trafficking dynamics of the auxin efflux carrier PIN-FORMED2 involved in root tropic responses and meristem organization. Our work links the local action of secretory peptides with phytohormone transport., (Copyright © 2012 Elsevier Inc. All rights reserved.)

• The seasonal timing of growth events is crucial to tree distribution and conservation. The seasonal growth cycle is strongly adapted to the local climate that is changing because of global warming. We studied bud set as one cornerstone of the seasonal growth cycle in an integrative approach. • Bud set was dissected at the phenotypic level into several components, and phenotypic components with most genetic variation were identified. While phenotypic variation resided in the timing of growth cessation, and even so more in the duration from growth cessation to bud set, the timing of growth cessation had a stronger genetic component in both natural and hybrid populations. • Quantitative trait loci (QTL) were identified for the most discriminative phenotypic bud-set components across four poplar pedigrees. The QTL from different pedigrees were recurrently detected in six regions of the poplar genome. • These regions of 1.83-4.25 Mbp in size, containing between 202 and 394 genes, form the basis for further molecular-genetic dissection of bud set., (© The Authors (2010). Journal compilation © New Phytologist Trust (2010).)

Lignin engineering is a promising strategy to optimize lignocellulosic plant biomass for use as a renewable feedstock for agro-industrial applications. Current efforts focus on engineering lignin with monomers that are not normally incorporated into wild-type lignins. Here we describe an Arabidopsis line in which the lignin is derived to a major extent from a non-traditional monomer. The combination of mutation in the gene encoding caffeic acid O-methyltransferase (comt) with over-expression of ferulate 5-hydroxylase under the control of the cinnamate 4-hydroxylase promoter (C4H:F5H1) resulted in plants with a unique lignin comprising almost 92% benzodioxane units. In addition to biosynthesis of this particular lignin, the comt C4H:F5H1 plants revealed massive shifts in phenolic metabolism compared to the wild type. The structures of 38 metabolites that accumulated in comt C4H:F51 plants were resolved by mass spectral analyses, and were shown to derive from 5-hydroxy-substituted phenylpropanoids. These metabolites probably originate from passive metabolism via existing biochemical routes normally used for 5-methoxylated and 5-unsubstituted phenylpropanoids and from active detoxification by hexosylation. Transcripts of the phenylpropanoid biosynthesis pathway were highly up-regulated in comt C4H:F5H1 plants, indicating feedback regulation within the pathway. To investigate the role of flavonoids in the abnormal growth of comt C4H:F5H1 plants, a mutation in a gene encoding chalcone synthase (chs) was crossed in. The resulting comt C4H:F5H1 chs plants showed partial restoration of growth. However, a causal connection between flavonoid deficiency and this restoration of growth was not demonstrated; instead, genetic interactions between phenylpropanoid and flavonoid biosynthesis could explain the partial restoration. These genetic interactions must be taken into account in future cell-wall engineering strategies., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula x Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

The growth of perennial plants in the temperate zone alternates with periods of dormancy that are typically initiated during bud development in autumn. In a systems biology approach to unravel the underlying molecular program of apical bud development in poplar (Populus tremula x Populus alba), combined transcript and metabolite profiling were applied to a high-resolution time course from short-day induction to complete dormancy. Metabolite and gene expression dynamics were used to reconstruct the temporal sequence of events during bud development. Importantly, bud development could be dissected into bud formation, acclimation to dehydration and cold, and dormancy. To each of these processes, specific sets of regulatory and marker genes and metabolites are associated and provide a reference frame for future functional studies. Light, ethylene, and abscisic acid signal transduction pathways consecutively control bud development by setting, modifying, or terminating these processes. Ethylene signal transduction is positioned temporally between light and abscisic acid signals and is putatively activated by transiently low hexose pools. The timing and place of cell proliferation arrest (related to dormancy) and of the accumulation of storage compounds (related to acclimation processes) were established within the bud by electron microscopy. Finally, the identification of a large set of genes commonly expressed during the growth-to-dormancy transitions in poplar apical buds, cambium, or Arabidopsis thaliana seeds suggests parallels in the underlying molecular mechanisms in different plant organs.

Genetical metabolomics [metabolite profiling combined with quantitative trait locus (QTL) analysis] has been proposed as a new tool to identify loci that control metabolite abundances. This concept was evaluated in a case study with the model tree Populus. Using HPLC, the peak abundances were analyzed of 15 closely related flavonoids present in apical tissues of two full-sib poplar families, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24, and correlation and QTL analysis were used to detect flux control points in flavonoid biosynthesis. Four robust metabolite quantitative trait loci (mQTL), associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of these mQTL, and the corresponding candidate genes were mapped. The data indicate that the combination of metabolite profiling with QTL analysis is a valuable tool to identify control points in a complex metabolic pathway of closely related compounds.

Populus deltoides, P. nigra, and P. trichocarpa are the most important species for poplar breeding programs worldwide. In addition, Populus has become a model for fundamental research on trees. Linkage maps were constructed for these three species by analyzing progeny of two controlled crosses sharing the same female parent, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24. The two-way pseudotestcross mapping strategy was used to construct the maps. Amplified fragment length polymorphism (AFLP) markers that segregated 1:1 were used to form the four parental maps. Microsatellites and sequence-tagged sites were used to align homoeologous groups between the maps and to merge linkage groups within the individual maps. Linkage analysis and alignment of the homoeologous groups resulted in 566 markers distributed over 19 groups for P. deltoides covering 86% of the genome, 339 markers distributed over 19 groups for P. trichocarpa covering 73%, and 369 markers distributed over 28 groups for P. nigra covering 61%. Several tests for randomness showed that the AFLP markers were randomly distributed over the genome.

Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.