Exploring the Seasonal Dynamics and Molecular Mechanism of Wood Formation in Gymnosperm Trees.

Forests, comprising 31% of the Earth's surface, play pivotal roles in regulating the carbon, water, and energy cycles. Despite being far less diverse than angiosperms, gymnosperms account for over 50% of the global woody biomass production. To sustain growth and development, gymnosperms have evolved the capacity to sense and respond to cyclical environmental signals, such as changes in photoperiod and seasonal temperature, which initiate growth (spring and summer) and dormancy (fall and winter). Cambium, the lateral meristem responsible for wood formation, is reactivated through a complex interplay among hormonal, genetic, and epigenetic factors. Temperature signals perceived in early spring induce the synthesis of several phytohormones, including auxins, cytokinins, and gibberellins, which in turn reactivate cambium cells. Additionally, microRNA-mediated genetic and epigenetic pathways modulate cambial function. As a result, the cambium becomes active during the summer, resulting in active secondary xylem (i.e., wood) production, and starts to become inactive in autumn. This review summarizes and discusses recent findings regarding the climatic, hormonal, genetic, and epigenetic regulation of wood formation in gymnosperm trees (i.e., conifers) in response to seasonal changes.

Embryogenic Stem Cell Identity after Protoplast Isolation from Daucus carota and Recovery of Regeneration Ability through Protoplast Culture.

Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 106 protoplasts·g−1 FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 104 protoplasts·g−1 FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6−6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.

Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA.

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar.

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1-sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Heparin-Mimicking Polymer-Based In Vitro Platform Recapitulates In Vivo Muscle Atrophy Phenotypes.

The cell-cell/cell-matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.

Overexpression of a Poplar RING-H2 Zinc Finger, Ptxerico, Confers Enhanced Drought Tolerance via Reduced Water Loss and Ion Leakage in Populus.

Drought stress is one of the major environmental problems in the growth of crops and woody perennials, but it is getting worse due to the global climate crisis. XERICO, a RING (Really Interesting New Gene) zinc-finger E3 ubiquitin ligase, has been shown to be a positive regulator of drought tolerance in plants through the control of abscisic acid (ABA) homeostasis. We characterized a poplar (Populus trichocarpa) RING protein family and identified the closest homolog of XERICO called PtXERICO. Expression of PtXERICO is induced by both salt and drought stress, and by ABA treatment in poplars. Overexpression of PtXERICO in Arabidopsis confers salt and ABA hypersensitivity in young seedlings, and enhances drought tolerance by decreasing transpirational water loss. Consistently, transgenic hybrid poplars overexpressing PtXERICO demonstrate enhanced drought tolerance with reduced transpirational water loss and ion leakage. Subsequent upregulation of genes involved in the ABA homeostasis and drought response was confirmed in both transgenic Arabidopsis and poplars. Taken together, our results suggest that PtXERICO will serve as a focal point to improve drought tolerance of woody perennials.

Wood forming tissue-specific bicistronic expression of PdGA20ox1 and PtrMYB221 improves both the quality and quantity of woody biomass production in a hybrid poplar.

With the exponential growth of the human population and industrial developments, research on renewable energy resources is required to alleviate environmental and economic impacts caused by the consumption of fossil fuels. In this study, we present a synthetic biological application of a wood forming tissue-specific bicistronic gene expression system to improve both the quantity and quality of woody biomass to minimize undesirable growth penalties. Our transgenic poplars, designed to express both PdGA20ox1 (a GA20-oxidase from Pinus densiflora producing bioactive gibberellin, GA) and PtrMYB221 (a MYB transcription factor negatively regulating lignin biosynthesis) under the developing xylem (DX) tissue-specific promoter (i.e., DX15::PdGA20ox1-2A-PtrMYB221 poplar), resulted in a 2-fold increase in biomass quantity compared to wild-type (WT), without undesirable growth defects. A similar phenotype was observed in transgenic Arabidopsis plants harboring the same gene constructs. These phenotypic consequences were further verified in the field experiments. Importantly, our transgenic poplars exhibited an improved quality of biomass with reduced lignin content (~16.0 wt%) but increased holocellulose content (~6.6 wt%). Furthermore, the saccharification efficiency of our transgenic poplar increased significantly by up to 8%. Our results demonstrate that the controlled production of both GA and a secondary wall modifying regulator in the same spatio-temporal manner can be utilized as an efficient biotechnological tool for producing the desired multi-purpose woody biomass.

Transcriptomic Analysis of Kalopanax septemlobus and Characterization of KsBAS, CYP716A94 and CYP72A397 Genes Involved in Hederagenin Saponin Biosynthesis.

Kalopanax septemlobus, commonly named the castor aralia tree, is a highly valued woody medicinal tree belonging to the family Araliaceae. Kalopanax septemlobus contains approximately 15 triterpenoid saponins primarily constituted of hederagenin aglycones. Hederagenin is a representative precursor for hemolytic saponin in plants. In the present study, transcriptome analysis was performed to discover genes involved in hederagenin saponin biosynthesis in K. septemlobus. De novo assembly generated 82,698 unique sequences, including 17,747 contigs and 64,951 singletons, following 454 pyrosequencing. Oxidosqualene cyclases (OSCs) are enzymes that catalyze the formation of diverse triterpene skeletons from 2,3-oxidosqualene. Heterologous expression of an OSC sequence in yeast revealed that KsBAS is a ß-amyrin synthase gene. Cytochrome P450 genes (CYPs) make up a supergene family in the plant genome and play a key role in the biosynthesis of sapogenin aglycones. In total, 95 contigs and 110 singletons annotated as CYPs were obtained by sequencing the K. septemlobus transcriptome. By heterologous expression in yeast, we found that CYP716A94 was ß-amyrin 28-oxidase involved in oleanolic acid production from ß-amyrin, and CYP72A397 was oleanolic acid 23-hydroxylase involved in hederagenin production from oleanolic acid. Engineered yeast co-expressing KsBAS, CYP716A94 and CYP72A397 produced hederagenin. Kalopanax septemlobus CYP72A397 is a novel CYP enzyme that synthesizes hederagenin aglycone from oleanolic acid as a single product. In conclusion, we characterized three genes participating in sequential steps for hederagenin biosynthesis from ß-amyrin, which are likely to play a major role in hederagenin saponin biosynthesis in K. septemlobus.

Gain-of-function mutation of AtDICE1, encoding a putative endoplasmic reticulum-localized membrane protein, causes defects in anisotropic cell elongation by disturbing cell wall integrity in Arabidopsis.

Background and Aims: Anisotropic cell elongation depends on cell wall relaxation and cellulose microfibril arrangement. The aim of this study was to characterize the molecular function of AtDICE1 encoding a novel transmembrane protein involved in anisotropic cell elongation in Arabidopsis. Methods: Phenotypic characterizations of transgenic Arabidopsis plants mis-regulating AtDICE1 expression with different pharmacological treatments were made, and biochemical, cell biological and transcriptome analyses were performed. Key Results: Upregulation of AtDICE1 in Arabidopsis (35S::AtDICE1) resulted in severe dwarfism, probably caused by defects in anisotropic cell elongation. Epidermal cell swelling was evident in all tissues, and abnormal secondary wall thickenings were observed in pith cells of stems. These phenotypes were reproduced not only by inducible expression of AtDICE1 but also by overexpression of its poplar homologue in Arabidopsis. RNA interference suppression lines of AtDICE1 resulted in no observable phenotypic changes. Interestingly, wild-type plants treated with isoxaben, a cellulose biosynthesis inhibitor, phenocopied the 35S::AtDICE1 plants, suggesting that cellulose biosynthesis was compromised in the 35S::AtDICE1 plants. Indeed, disturbed cortical microtubule arrangements in 35S::AtDICE1/GFP-TuA6 plants were observed, and the cellulose content was significantly reduced in 35S::AtDICE1 plants. A promoter::GUS analysis showed that AtDICE1 is mainly expressed in vascular tissue, and transient expression of GFP:AtDICE1 in tobacco suggests that AtDICE1 is probably localized in the endoplasmic reticulum (ER). In addition, the external N-terminal conserved domain of AtDICE1 was found to be necessary for AtDICE1 function. Whole transcriptome analyses of 35S::AtDICE1 revealed that many genes involved in cell wall modification and stress/defence responses were mis-regulated. Conclusions: AtDICE1, a novel ER-localized transmembrane protein, may contribute to anisotropic cell elongation in the formation of vascular tissue by affecting cellulose biosynthesis.

Transcriptomic analysis of Siberian ginseng (Eleutherococcus senticosus) to discover genes involved in saponin biosynthesis.

BACKGROUND: Eleutherococcus senticosus, Siberian ginseng, is a highly valued woody medicinal plant belonging to the family Araliaceae. E. senticosus produces a rich variety of saponins such as oleanane-type, noroleanane-type, 29-hydroxyoleanan-type, and lupane-type saponins. Genomic or transcriptomic approaches have not been used to investigate the saponin biosynthetic pathway in this plant. RESULT: In this study, de novo sequencing was performed to select candidate genes involved in the saponin biosynthetic pathway. A half-plate 454 pyrosequencing run produced 627,923 high-quality reads with an average sequence length of 422 bases. De novo assembly generated 72,811 unique sequences, including 15,217 contigs and 57,594 singletons. Approximately 48,300 (66.3%) unique sequences were annotated using BLAST similarity searches. All of the mevalonate pathway genes for saponin biosynthesis starting from acetyl-CoA were isolated. Moreover, 206 reads of cytochrome P450 (CYP) and 145 reads of uridine diphosphate glycosyltransferase (UGT) sequences were isolated. Based on methyl jasmonate (MeJA) treatment and real-time PCR (qPCR) analysis, 3 CYPs and 3 UGTs were finally selected as candidate genes involved in the saponin biosynthetic pathway. CONCLUSIONS: The identified sequences associated with saponin biosynthesis will facilitate the study of the functional genomics of saponin biosynthesis and genetic engineering of E. senticosus.

The poplar basic helix-loop-helix transcription factor BEE3 - Like gene affects biomass production by enhancing proliferation of xylem cells in poplar.

Brassinosteroids (BRs) play important roles in many aspects of plant growth and development, including regulation of vascular cambium activities and cell elongation. BR-induced BEE3 (brassinosteroid enhanced expression 3) is required for a proper BR response. Here, we identified a poplar (Populus alba × Populus glandulosa) BEE3-like gene, PagBEE3L, encoding a putative basic helix-loop-helix (bHLH)-type transcription factor. Expression of PagBEE3L was induced by brassinolide (BL). Transcripts of PagBEE3L were mainly detected in stems, with the internode having a low level of transcription and the node having a relatively higher level. The function of the PagBEE3L gene was investigated through phenotypic analyses with PagBEE3L-overexpressing (ox) transgenic lines. This work particularly focused on a potential role of PagBEE3L in stem growth and development of polar. The PagBEE3L-ox poplar showed thicker and longer stems than wild-type plants. The xylem cells from the stems of PagBEE3L-ox plants revealed remarkably enhanced proliferation, resulting in an earlier thickening growth than wild-type plants. Therefore, this work suggests that xylem development of poplar is accelerated in PagBEE3L-ox plants and PagBEE3L plays a role in stem growth by increasing the proliferation of xylem cells to promote the initial thickening growth of poplar stems.

Seasonal Developing Xylem Transcriptome Analysis of Pinus densiflora Unveils Novel Insights for Compression Wood Formation.

Wood is the most important renewable resource not only for numerous practical utilizations but also for mitigating the global climate crisis by sequestering atmospheric carbon dioxide. The compressed wood (CW) of gymnosperms, such as conifers, plays a pivotal role in determining the structure of the tree through the reorientation of stems displaced by environmental forces and is characterized by a high content of lignin. Despite extensive studies on many genes involved in wood formation, the molecular mechanisms underlying seasonal and, particularly, CW formation remain unclear. This study examined the seasonal dynamics of two wood tissue types in Pinus densiflora: CW and opposite wood (OW). RNA sequencing of developing xylem for two consecutive years revealed comprehensive transcriptome changes and unique differences in CW and OW across seasons. During growth periods, such as spring and summer, we identified 2255 transcripts with differential expression in CW, with an upregulation in lignin biosynthesis genes and significant downregulation in stress response genes. Notably, among the laccases critical for monolignol polymerization, PdeLAC17 was found to be specifically expressed in CW, suggesting its vital role in CW formation. PdeERF4, an ERF transcription factor preferentially expressed in CW, seems to regulate PdeLAC17 activity. This research provides an initial insight into the transcriptional regulation of seasonal CW development in P. densiflora, forming a foundation for future studies to enhance our comprehension of wood formation in gymnosperms.

Comparative functional analysis of PdeNAC2 and AtVND6 in the tracheary element formation.

Tracheary elements (i.e. vessel elements and tracheids) are highly specialized, non-living cells present in the water-conducting xylem tissue. In angiosperms, proteins in the VASCULAR-RELATED NAC-DOMAIN (VND) subgroup of the NAC (NAM, ATAF1,2, and CUC2) transcription factor family (e.g. AtVND6) are required for the differentiation of vessel elements through transcriptional regulation of genes responsible for secondary cell wall formation and programmed cell death. Gymnosperms, however, produce only tracheids, the mechanism of which remains elusive. Here, we report functional characteristics of PdeNAC2, a VND homolog in Pinus densiflora, as a key regulator of tracheid formation. Interestingly, our molecular genetic analyses show that PdeNAC2 can induce the formation of vessel element-like cells in angiosperm plants, demonstrated by transgenic overexpression of either native or NAC domain-swapped synthetic genes of PdeNAC2 and AtVND6 in both Arabidopsis and hybrid poplar. Subsequently, genome-wide identification of direct target (DT) genes of PdeNAC2 and AtVND6 revealed 138 and 174 genes as putative DTs, respectively, but only 17 genes were identified as common DTs. Further analyses have found that PdeNAC2 does not control some AtVND6-dependent vessel differentiation genes in angiosperm plants, such as AtVRLK1, LBD15/30 and pit-forming Rho-like GTPases from plant (ROP) signaling genes. Collectively, our results suggest that different target gene repertoires of PdeNAC2 and AtVND6 may contribute to the evolution of tracheary elements.

Current Understanding of the Genetics and Molecular Mechanisms Regulating Wood Formation in Plants.

Unlike herbaceous plants, woody plants undergo volumetric growth (a.k.a. secondary growth) through wood formation, during which the secondary xylem (i.e., wood) differentiates from the vascular cambium. Wood is the most abundant biomass on Earth and, by absorbing atmospheric carbon dioxide, functions as one of the largest carbon sinks. As a sustainable and eco-friendly energy source, lignocellulosic biomass can help address environmental pollution and the global climate crisis. Studies of Arabidopsis and poplar as model plants using various emerging research tools show that the formation and proliferation of the vascular cambium and the differentiation of xylem cells require the modulation of multiple signals, including plant hormones, transcription factors, and signaling peptides. In this review, we summarize the latest knowledge on the molecular mechanism of wood formation, one of the most important biological processes on Earth.

Knockdown of PagSAP11 Confers Drought Resistance and Promotes Lateral Shoot Growth in Hybrid Poplar (Populus alba × Populus tremula var. glandulosa).

Plants have evolved defense mechanisms to overcome unfavorable climatic conditions. The growth and development of plants are regulated in response to environmental stress. In this study, we investigated the molecular and physiological characteristics of a novel gene PagSAP11 in hybrid poplar (Populus alba × Populus tremula var. glandulosa) under drought stress. PagSAP11, a stress-associated protein (SAP) family gene, encodes a putative protein containing an A20 and AN1 zinc-finger domain at its N- and C-termini, respectively. Knockdown of PagSAP11 transgenic poplars (SAP11-Ri) enhanced their tolerance to drought stress compared with wild type plants. Moreover, the RNAi lines showed increased branching of lateral shoots that led to a gain in fresh weight, even when grown in the living modified organism (LMO) field. In SAP11-Ri transgenic plants, the expression levels of genes involved in axillary bud outgrowth and cell proliferation such as DML10, CYP707A and RAX were increased while the DRM gene which involved in bud dormancy was down-regulated. Taken together, these results indicate that PagSAP11 represents a promising candidate gene for engineering trees with improved stress tolerance and growth during unfavorable conditions.

Establishment of an Efficient In Vitro Propagation of Cnidium officinale Makino and Selection of Superior Clones through Flow Cytometric Assessment of DNA Content.

Cnidium officinale is a valuable medicinal plant cultivated in Asia for its rhizomes. This study reports the in vitro regeneration of Cnidium officinale plants and the induction of rhizomes from microshoots. The rhizomatous buds of Cnidium officinale induced multiple shoots on Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 BA, which led to the regeneration of plants within four weeks of culture. After four weeks of culture, the plants were assessed for fresh weight, the number of leaves, the number of roots, and the length of roots to compare the performance of the different clones. The clones with good growth characteristics were selected with the aid of a flow cytometric analysis of 2C nuclear DNA content. The plants bearing high DNA values showed better growth characteristics. Various factors, namely, sucrose concentration (30, 50, 70, and 90 g L-1), ABA (0, 0.5, 1.0, and 2.0 mg L-1), the synergistic effects of BA (1.0 mg L-1) + NAA (0.5 mg L-1) and BA (1.0 mg L-1) + NAA (0.5 mg L-1) + ABA (1.0 mg L-1) with or without activated charcoal (1 g L-1), and light and dark incubation were tested on rhizome formation from microshoots. The results of the above experiments suggest that MS medium supplemented with 50 g L-1 sucrose, 1.0 mg L-1 ABA, and 1 g L-1 AC is good for the induction of rhizomes from the shoots of Cnidium officinale. Plantlets with rhizomes were successfully transferred to pots, and they showed 100% survival.

Wood transcriptome analysis of Pinus densiflora identifies genes critical for secondary cell wall formation and NAC transcription factors involved in tracheid formation.

Although conifers have significant ecological and economic value, information on transcriptional regulation of wood formation in conifers is still limited. Here, to gain insight into secondary cell wall (SCW) biosynthesis and tracheid formation in conifers, we performed wood tissue-specific transcriptome analyses of Pinus densiflora (Korean red pine) using RNA sequencing. In addition, to obtain full-length transcriptome information, PacBio single molecule real-time iso-sequencing was carried out using RNAs from 28 tissues of P. densiflora. Subsequent comparative tissue-specific transcriptome analysis successfully pinpointed critical genes encoding key proteins involved in biosynthesis of the major secondary wall components (cellulose, galactoglucomannan, xylan and lignin). Furthermore, we predicted a total of 62 NAC (NAM, ATAF1/2 and CUC2) family transcription factor members and identified seven PdeNAC genes preferentially expressed in developing xylem tissues in P. densiflora. Protoplast-based transcriptional activation analysis found that four PdeNAC genes, homologous to VND, NST and SND/ANAC075, upregulated GUS activity driven by an SCW-specific cellulose synthase promoter. Consistently, transient overexpression of the four PdeNACs induced xylem vessel cell-like SCW deposition in both tobacco (Nicotiana benthamiana) and Arabidopsis leaves. Taken together, our data provide a foundation for further research to unravel transcriptional regulation of wood formation in conifers, especially SCW formation and tracheid differentiation.

A Novel Robot-Assisted Kinematic Measure for Children with Attention-Deficit/Hyperactivity Disorder: A Preliminary Study.

OBJECTIVE: Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, and impulsivity. In contrast to neurocognitive measurements of inattention and impulsivity, there has been limited research regarding the objective measurement of hyperactivity in youths with ADHD. The purpose of the present study was to investigate the clinical effectiveness of a newly developed Robot-assisted Kinematic Measure for ADHD (RAKMA) in children with ADHD. METHODS: In total, 35 children with ADHD aged 5 to 12 years and 50 healthy controls (HCs) were recruited, and the parents completed the Child Behavior Checklist and the Korean ADHD Diagnostic Scale. RAKMA performance was represented by RAKMA stimulus-response and hyperactivity variables. We compared the RAKMA performance of those with ADHD and with that of HCs and also investigated the correlation between the RAKMA variables and ADHD clinical scale scores. RESULTS: Significant differences between the ADHD and HC groups were observed regarding most RAKMA variables, including correct reactions, commission errors, omission errors, reaction times, migration distance, and migration speed scores. Significant correlations were detected between various ADHD clinical scale scores and RAKMA variables. CONCLUSION: The RAKMA was a clinically useful tool for objectively measuring hyperactivity symptoms in children with ADHD. Further studies with larger samples are warranted.

PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathway in a hybrid poplar.

Both anthocyanins and lignins are essential secondary metabolites in plant growth and development. Their biosynthesis is metabolically interconnected and diverges in the central metabolite 4-coumaroyl CoA of the phenylpropanoid pathway. Considerable progress has been made in understanding transcriptional regulation of genes involved in lignin and anthocyanin synthesis pathways, but the concerted regulation of these pathways is not yet fully understood. Here, we functionally characterized PtrMYB120, a R2R3-MYB transcription factor from Populus trichocarpa. Overexpression of PtrMYB120 in a hybrid poplar (i.e., 35S::PtrMYB120) was associated with increased anthocyanin (i.e., cyanidin 3-O-glucoside) accumulation and upregulation of anthocyanin biosynthetic genes. However, transgenic poplars with dominant suppression of PtrMYB120 function achieved by fusing the ERF-associated amphiphilic repression motif to PtrMYB120 (i.e., 35S::PtrMYB120-SRDX) had a dramatic decrease in not only anthocyanin but also Klason lignin content with downregulation of both anthocyanin and lignin biosynthetic genes. Indeed, 35S::PtrMYB120-SRDX poplars had irregularly shaped xylem vessels with reduced S-lignin content in stems, which was proportionally related to the level of the introduced PtrMYB120-SRDX gene. Furthermore, protoplast-based transcriptional activation assay using the PtrMYB120-GR system suggested that PtrMYB120 directly regulates genes involved in both anthocyanin and lignin biosynthesis, including chalcone synthase and ferulate-5 hydroxylase. Interestingly, the saccharification efficiency of line #6 of 35S::PtrMYB120-SRDX poplars, which had slightly reduced lignin content with a normal growth phenotype, was dramatically enhanced (>45%) by NaOH treatment. Taken together, our results suggest that PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathways and can be targeted to enhance saccharification efficiency in woody perennials.

Large-scale production of recombinant miraculin protein in transgenic carrot callus suspension cultures using air-lift bioreactors.

Miraculin, derived from the miracle fruit (Synsepalum dulcificum), is a taste-regulating protein that interacts with human sweet-taste receptors and transforms sourness into sweet taste. Since miracle fruit is cultivated in West Africa, mass production of miraculin is limited by regional and seasonal constraints. Here, we investigated mass production of recombinant miraculin in carrot (Daucus carota L.) callus cultures using an air-lift bioreactor. To increase miraculin expression, the oxidative stress-inducible SWPA2 promoter was used to drive the expression of miraculin gene under various stress treatments. An 8 h treatment of hydrogen peroxide (H2O2) and salt (NaCl) increased the expression of miraculin gene by fivefold compared with the untreated control. On the other hand, abscisic acid, salicylic acid, and methyl jasmonate treatments showed no significant impact on miraculin gene expression compared with the control. This shows that since H2O2 and NaCl treatments induce oxidative stress, they activate the SWPA2 promoter and consequently up-regulate miraculin gene expression. Thus, the results of this study provide a foundation for industrial-scale production of recombinant miraculin protein using transgenic carrot cells as a heterologous host.