Jasmonate activates secondary cell wall biosynthesis through MYC2-MYB46 module.

Formation of secondary cell wall (SCW) is tightly regulated spatiotemporally by various developmental and environmental signals. Successful fine-tuning of the trade-off between SCW biosynthesis and stress responses requires a better understanding of how plant growth is regulated under environmental stress conditions. However, the current understanding of the interplay between environmental signaling and SCW formation is limited. The lipid-derived plant hormone jasmonate (JA) and its derivatives are important signaling components involved in various physiological processes including plant growth, development, and abiotic/biotic stress responses. Recent studies suggest that JA is involved in SCW formation but the signaling pathway has not been studied for how JA regulates SCW formation. We tested this hypothesis using the transcription factor MYB46, a master switch for SCW biosynthesis, and JA treatments. Both the transcript and protein levels of MYB46, a master switch for SCW formation, were significantly increased by JA treatment, resulting in the upregulation of SCW biosynthesis. We then show that this JA-induced upregulation of MYB46 is mediated by MYC2, a central regulator of JA signaling, which binds to the promoter of MYB46. We conclude that this MYC2-MYB46 module is a key component of the plant response to JA in SCW formation.

Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

Mitogen-activated protein kinase 6 negatively regulates secondary wall biosynthesis by modulating MYB46 protein stability in Arabidopsis thaliana.

The R2R3-MYB transcription factor MYB46 functions as a master switch for secondary cell wall biosynthesis, ensuring the exquisite expression of the secondary wall biosynthetic genes in the tissues where secondary walls are critical for growth and development. At the same time, suppression of its function is needed when/where formation of secondary walls is not desirable. Little is known about how this opposing control of secondary cell wall formation is achieved. We used both transient and transgenic expression of MYB46 and mitogen-activated protein kinase 6 (MPK6) to investigate the molecular mechanism of the post-translational regulation of MYB46. We show that MYB46 is phosphorylated by MPK6, leading to site specific phosphorylation-dependent degradation of MYB46 by the ubiquitin-mediated proteasome pathway. In addition, the MPK6-mediated MYB46 phosphorylation was found to regulate in planta secondary wall forming function of MYB46. Furthermore, we provide experimental evidences that MYB83, a paralog of MYB46, is not regulated by MPK6. The coupling of MPK signaling to MYB46 function provides insights into the tissue- and/or condition-specific activity of MYB46 for secondary wall biosynthesis.

SNF1-related protein kinase 1 represses Arabidopsis growth through post-translational modification of E2Fa in response to energy stress.

Cellular sugar starvation and/or energy deprivation serves as an important signaling cue for the live cells to trigger the necessary stress adaptation response. When exposed to cellular energy stress (ES) conditions, the plants reconfigure metabolic pathways and rebalance energy status while restricting vegetative organ growth. Despite the vital importance of this ES-induced growth restriction, the regulatory mechanism underlying the response remains largely elusive in plants. Using plant cell- and whole plant-based functional analyses coupled with extended genetic validation, we show that cellular ES-activated SNF1-related protein kinase 1 (SnRK1.1) directly interacts with and phosphorylates E2Fa transcription factor, a critical cell cycle regulator. Phosphorylation of E2Fa by SnRK1.1 leads to its proteasome-mediated protein degradation, resulting in S-phase repression and organ growth restriction. Our findings show that ES-dependently activated SnRK1.1 adjusts cell proliferation and vegetative growth for plants to cope with constantly fluctuating environments.

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.

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.

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.

Development of bisphenol A (BPA)-sensing indicator Arabidopsis thaliana which synthesizes anthocyanin in response to BPA in leaves.

Bisphenol A (BPA) is an estrogenic endocrine disruptor which disturbs a normal animal development. We generated an indicator plant that senses and provides a clear visual indicator of an estrogen-like compound BPA in the environment. We developed transgenic Arabidopsis lines expressing a construct designed to synthesize anthocyanin (thus showing a red color) in response to BPA. We transformed Arabidopsis with a recombinant vector containing the chimeric estrogen receptor (XVE region), LAP and coding region of PtrMYB119 (transcription factor involved in anthocyanin biosynthesis in poplar and Arabidopsis). Upon binding of the estrogen compound to the ligand-binding domain of E (estrogen receptor) in XVE, the XV domain binds to LAP promoter and triggering the transcription of PtrMYB119 with a subsequent enhancement of anthocyanin biosynthetic gene expression, resulting in anthocyanin synthesis. The leaves of the transgenic Arabidopsis line XVE-PtrMYB119 turned red in the presence of 10 ppm BPA. The transcript level of PtrMYB119 peaked at day 3 of BPA exposure, then decreased to its minimal level at day 5. Similar expression patterns to that of PtrMYB119 were detected for genes encoding the anthocyanin biosynthetic enzymes chalcone synthase, chalcone flavanone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and UFGT (UGT78D2). The leaves of transgenic plants did not turn red in response to BPA at concentrations below 10 ppm, but PtrMYB119 expression was induced by BPA at concentrations as low as 1 ppt BPA. Since this transgenic plant turns red in the presence of BPA without any experimental procedures, this line can be easily used by non-scientists.

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.

Developing xylem-preferential expression of PdGA20ox1, a gibberellin 20-oxidase 1 from Pinus densiflora, improves woody biomass production in a hybrid poplar.

Woody biomass has gained popularity as an environmentally friendly, renewable and sustainable resource for liquid fuel production. Here, we demonstrate biotechnological improvement of the quantity and quality of woody biomass by employing developing xylem (DX)-preferential production of gibberellin (GA), a phytohormone that positively regulates stem growth. First, for the proof of concept experiment, we produced transgenic Arabidopsis plants expressing GA20-oxidase, a key enzyme in the production of bioactive GAs, from Pinus densiflora (PdGA20ox1) under the control of either a constitutive 35S promoter, designated 35S::PdGA20ox1, or a DX-specific promoter (originated from poplar), designated DX15::PdGA20ox1. As we hypothesized, both transgenic Arabidopsis plants (35S::PdGA20ox1 and DX15::PdGA20ox1) exhibited an accelerated stem growth that resulted in a large increase of biomass, up to 300% compared to wild-type control plants, together with increased secondary wall thickening and elongation of fibre cells. Next, we applied our concept to the production of transgenic poplar trees. Both transgenic poplar trees (35S::PdGA20ox1 and DX15::PdGA20ox1) showed dramatic increases in biomass, up to 300%, with accelerated stem growth and xylem differentiation. Cell wall monosaccharide composition analysis revealed that in both Arabidopsis and poplar, glucose and xylose contents were significantly increased. However, undesirable phenotypes of 35S::PdGA20ox1 poplar, including poor root growth and leaf development, were found. Interestingly, DX15::PdGA20ox1 poplar resulted in a reduction of undesirable phenotypes. Our results indicate that the controlled production of GAs through a tissue-specific promoter can be utilized as an efficient biotechnological tool for producing enhanced plant biomass, minimizing unwanted effects.

AtC3H14, a plant-specific tandem CCCH zinc-finger protein, binds to its target mRNAs in a sequence-specific manner and affects cell elongation in Arabidopsis thaliana.

AtC3H14 (At1 g66810) is a plant-specific tandem CCCH zinc-finger (TZF) protein that belongs to the 68-member CCCH family in Arabidopsis thaliana. In animals, TZFs have been shown to bind and recruit target mRNAs to the cytoplasmic foci where mRNA decay enzymes are active. However, it is not known whether plant TZF proteins such as AtC3H14 function. So far, no mRNA targets of plant TZFs have been identified. We have obtained several lines of experimental evidence in support of our hypothesis that AtC3H14 is involved in post-transcriptional regulation of its target genes. Nucleic acid binding assays using [(35) S]-labeled AtC3H14 protein showed that AtC3H14 could bind to ssDNA, dsDNA, and ribohomopolymers, suggesting its RNA-binding activity. RNA immunoprecipitation (RIP) assay identified several putative target RNAs of AtC3H14, including a polygalacturonase, a well-known cell wall modifying gene. RNA electrophoretic mobility shift assays (RNA-EMSA) were used to confirm the RIP results and demonstrate that the TZF domain of AtC3H14 is required for the target RNA binding. Microarray analysis of 35S::AtC3H14 plants revealed that many of the cell wall elongation and/or modification-associated genes were differentially expressed, which is consistent with the cell elongation defect phenotype and the changes in the cell wall monosaccharide composition. In addition, yeast activation assay showed that AtC3H14 also function as a transcriptional activator, which is consistent with the previous finding that AtC3H14 activate the secondary wall biosynthesis genes. Taken together, we conclude that AtC3H14 may play a key role in both transcriptional and post-transcriptional regulation.

MYB46 directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis.

Cellulose is the most abundant biopolymer on Earth. Three cellulose synthases (CESA4, CESA7 and CESA8) are necessary for cellulose production in the secondary cell walls of Arabidopsis. Little is known about how expression of these CESA genes is regulated. We recently identified a cis-regulatory element (M46RE) that is recognized by MYB46, which is a master switch for secondary wall formation in Arabidopsis. A genome-wide survey of promoter sequences for the presence of M46REs led to the hypothesis that MYB46 may function as a direct regulator of all three secondary wall-associated cellulose synthase genes: CESA4, CESA7 and CESA8. We tested this hypothesis using several lines of experimental evidence. All three CESA genes are highly up-regulated by both constitutive and inducible over-expression of MYB46 in planta. Using a steroid receptor-based inducible activation system, we show that MYB46 directly activates transcription of the three CESA genes. We then used an electrophoretic mobility shift assay and chromatin immunoprecipitation analysis to confirm that MYB46 protein directly binds to the promoters of the three CESA genes both in vitro and in vivo. Furthermore, ectopic up-regulation of MYB46 resulted in a significant increase of crystalline cellulose content in Arabidopsis. Taken together, we have identified MYB46 as a transcription factor that directly regulates all three secondary wall-associated CESA genes. Yeast one-hybrid screening identified additional transcription factors that regulate the CESA genes. However, none of the putative regulators appears to be regulated by MYB46, suggesting the multi-faceted nature of transcriptional regulation of secondary wall cellulose biosynthesis.

Identification of direct targets of transcription factor MYB46 provides insights into the transcriptional regulation of secondary wall biosynthesis.

Secondary wall formation requires coordinated transcriptional regulation of the genes involved in the biosynthesis of the components of secondary wall. Transcription factor (TF) MYB46 (At5g12870) has been shown to function as a central regulator for secondary wall formation in Arabidopsis thaliana, activating biosynthetic genes as well as the TFs involved in the pathways. Recently, we reported that MYB46 directly regulates secondary wall-associated cellulose synthase (CESA4, CESA7, and CESA8) and a mannan synthase (CSLA9) genes. However, it is not known whether MYB46 directly activates the biosynthetic genes for hemicellulose and lignin, which are the other two major components of secondary wall. Based on the observations that the promoter regions of many of the secondary wall biosynthetic genes contain MYB46-binding cis-regulatory motif(s), we hypothesized that MYB46 directly regulates the genes involved in the biosynthesis of the secondary wall components. In this report, we describe several lines of experimental evidence in support of the hypothesis. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis showed that MYB46 directly binds to the promoters of 13 genes involved in lignin and xylan biosynthesis. We then used steroid receptor-based inducible activation system to confirm that MYB46 directly activates the transcription of the xylan and lignin biosynthetic genes. Furthermore, ectopic up-regulation of MYB46 resulted in a significant increase in xylose and a small increase in lignin content based on acetyl bromide soluble lignin measurements in Arabidopsis. Taken together, we conclude that MYB46 function as a central and direct regulator of the genes involved in the biosynthesis of all three major secondary wall components.

Gibberellin mediates the development of gelatinous fibres in the tension wood of inclined Acacia mangium seedlings.

BACKGROUND AND AIMS: Gibberellin stimulates negative gravitropism and the formation of tension wood in tilted Acacia mangium seedlings, while inhibitors of gibberellin synthesis strongly inhibit the return to vertical growth and suppress the formation of tension wood. To characterize the role of gibberellin in tension wood formation and gravitropism, this study investigated the role of gibberellin in the development of gelatinous fibres and in the changes in anatomical characteristics of woody elements in Acacia mangium seedlings exposed to a gravitational stimulus. METHODS: Gibberellin, paclobutrazol and uniconazole-P were applied to the soil in which seedlings were growing, using distilled water as the control. Three days after the start of treatment, seedlings were inclined at 45 ° to the vertical and samples were harvested 2 months later. The effects of the treatments on wood fibres, vessel elements and ray parenchyma cells were analysed in tension wood in the upper part of inclined stems and in the opposite wood on the lower side of inclined stems. KEY RESULTS: Application of paclobutrazol or uniconazole-P inhibited the increase in the thickness of gelatinous layers and prevented the elongation of gelatinous fibres in the tension wood of inclined stems. By contrast, gibberellin stimulated the elongation of these fibres. Application of gibberellin and inhibitors of gibberellin biosynthesis had only minor effects on the anatomical characteristics of vessel and ray parenchyma cells. CONCLUSIONS: The results suggest that gibberellin is important for the development of gelatinous fibres in the tension wood of A. mangium seedlings and therefore in gravitropism.

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.

ß-Glucosidase and Its Application in Bioconversion of Ginsenosides in Panax ginseng.

Ginsenosides are a group of bioactive compounds isolated from Panax ginseng. Conventional major ginsenosides have a long history of use in traditional medicine for both illness prevention and therapy. Bioconversion processes have the potential to create new and valuable products in pharmaceutical and biological activities, making them both critical for research and highly economic to implement. This has led to an increase in the number of studies that use major ginsenosides as a precursor to generate minor ones using ß-glucosidase. Minor ginsenosides may also have useful properties but are difficult to isolate from raw ginseng because of their scarcity. Bioconversion processes have the potential to create novel minor ginsenosides from the more abundant major ginsenoside precursors in a cost-effective manner. While numerous bioconversion techniques have been developed, an increasing number of studies have reported that ß-glucosidase can effectively and specifically generate minor ginsenosides. This paper summarizes the probable bioconversion mechanisms of two protopanaxadiol (PPD) and protopanaxatriol (PPT) types. Other high-efficiency and high-value bioconversion processes using complete proteins isolated from bacterial biomass or recombinant enzymes are also discussed in this article. This paper also discusses the various conversion and analysis methods and their potential applications. Overall, this paper offers theoretical and technical foundations for future studies that will be both scientifically and economically significant.

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.

Identification of a cis-acting regulatory motif recognized by MYB46, a master transcriptional regulator of secondary wall biosynthesis.

While many aspects of primary cell wall have been extensively elucidated, our current understanding of secondary wall biosynthesis is limited. Recently, transcription factor MYB46 has been identified as a master regulator of secondary wall biosynthesis in Arabidopsis thaliana. To gain better understanding of this MYB46-mediated transcriptional regulation, we analyzed the promoter region of a direct target gene, AtC3H14, of MYB46 and identified a cis-acting regulatory motif that is recognized by MYB46. This MYB46-responsive cis-regulatory element (M46RE) was further characterized and shown to have an eight-nucleotide core motif, RKTWGGTR. We used electrophoretic mobility shift assay, transient transcriptional activation assay and chromatin immunoprecipitation analysis to show that the M46RE was necessary and sufficient for MYB46-responsive transcription. Genome-wide analysis identified that the frequency of M46RE in the promoters were highly enriched among the genes upregulated by MYB46, especially in the group of genes involved in secondary wall biosynthesis.