Exogenous IAA application affects the specific characteristics of fluoranthene distribution in Arabidopsis.

Indole-3-acetic acid (IAA) is a crucial growth regulator involved in the accumulation of polycyclic aromatic hydrocarbons (PAHs). However, the precise physiological and molecular mechanisms underlying IAA-mediated plant growth and PAH accumulation are not yet fully understood. In this study, two distinct IAA-sensitive genotypes of Arabidopsis thaliana (wild type and Axr5 mutant) were chosen to investigate the mechanisms of fluoranthene (Flu) uptake and accumulation in plant tissues (roots and leaves) through physiological and molecular analyses. The results revealed that the Flu concentration in Axr5 leaves was significantly higher than that in wild-type (WT) leaves. In roots, the Flu content decreased significantly with increasing IAA treatment, while no significant changes were observed with lower IAA treatment. Principal component analysis demonstrated that Flu accumulation in Arabidopsis roots was associated with IAA concentrations, whereas Flu accumulation in leaves was dependent on the genotype. Moreover, Flu accumulation showed a positive correlation with the activity of glutathione S-transferase (GST) and root length and a positive correlation with catalase (CAT) and peroxidase (POD) activity in the leaves. Transcriptome analysis confirmed that the expression of the ethylene-related gene ATERF6 and GST-related genes ATGSTF14 and ATGSTU27 in roots, as well as the POD-related genes AtPRX9 and AtPRX25 and CAT-related gene AtCAT3 in leaves, played a role in Flu accumulation. Furthermore, WRKY transcription factors (TFs) in roots and NAC TFs in leaves were identified as important regulators of Flu accumulation. Understanding the mechanisms of Flu uptake and accumulation in A. thaliana provides valuable insights for regulating PAH accumulation in plants.

Five NUCLEAR FACTOR-Y subunit B genes in rapeseed (Brassica napus) promote flowering and root elongation in Arabidopsis.

MAIN CONCLUSION: Heterologous expression of BnNF-YB2, BnNF-YB3, BnNF-YB4, BnNF-YB5, or BnNF-YB6 from rapeseed promotes the floral process and also affects root development in Arabidopsis. The transcriptional regulator NUCLEAR FACTOR-Y (NF-Y) is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC proteins and is ubiquitous in yeast, animal, and plant systems. In this study, we found that five NF-YB proteins from rapeseed (Brassica napus), including BnNF-YB2, BnNF-YB3, BnNF-YB4, BnNF-YB5, and BnNF-YB6 (BnNF-YB2/3/4/5/6), all function in photoperiodic flowering and root elongation. Sequence alignment and phylogenetic analysis showed that BnNF-YB2/3 and BnNF-YB4/5/6 were clustered with Arabidopsis AtNF-YB2 and AtNF-YB3, respectively, implying that these NF-YBs are evolutionarily and functionally conserved. In support of this hypothesis, the heterologous expression of individual BnNF-YB2, 3, 4, 5, or 6 in Arabidopsis promoted early flowering under a long-day photoperiod. Further analysis suggested that BnNF-YB 2/3/4/5/6 elevated the expression of key downstream flowering time genes including CO, FT, LFY and SOC1. Promoter-GUS fusion analysis showed that the five BnNF-YBs were expressed in a variety of tissues at various developmental stages and GFP fusion analysis revealed that all BnNF-YBs were localized to the nucleus. In addition, we demonstrated that the heterologous expression of individual BnNF-YB2/3/4/5/6 in Arabidopsis promoted root elongation and increased the number of root tips formed under both normal and treatment with simulators of abiotic stress conditions.

Characterization of inulolytic enzymes from the Jerusalem artichoke-derived Glutamicibacter mishrai NJAU-1.

The rhizosphere context of inulin-accumulating plants, such as Jerusalem artichoke (Helianthus tuberosus), is an ideal starting basis for the discovery of inulolytic enzymes with potential for bio fructose production. We isolated a Glutamicibacter mishrai NJAU-1 strain from this context, showing exo-inulinase activity, releasing fructose from fructans. The growth conditions (pH 9.0; 15 °C) were adjusted, and the production of inulinase by Glutamicibacter mishrai NJAU-1 increased by 90% (0.32 U/mL). Intriguingly, both levan and inulin, but not fructose and sucrose, induced the production of exo-inulinase activity. Two exo-inulinase genes (inu1 and inu2) were cloned and heterologously expressed in Pichia pastoris. While INU2 preferentially hydrolyzed longer inulins, the smallest fructan 1-kestose appeared as the preferred substrate for INU1, also efficiently degrading nystose and sucrose. Active site docking studies with GFn- and Fn-type small inulins (G is glucose, F is fructose, and n is the number of ß (2-1) bound fructose moieties) revealed subtle substrate differences between INU1 and INU2. A possible explanation about substrate specificity and INU's protein structure is then suggested. KEY POINTS: • A Glutamicibacter mishrai strain harbored exo-inulinase activity. • Fructans induced the inulolytic activity in G. mishrai while the inulolytic activity was optimized at pH 9.0 and 15 °C. • Two exo-inulinases with differential substrate specificity were characterized.

Physiological and Transcriptional Responses of Industrial Rapeseed (Brassica napus) Seedlings to Drought and Salinity Stress.

Abiotic stress greatly inhibits crop growth and reduces yields. However, little is known about the transcriptomic changes that occur in the industrial oilseed crop, rapeseed (Brassica napus), in response to abiotic stress. In this study, we examined the physiological and transcriptional responses of rapeseed to drought (simulated by treatment with 15% (w/v) polyethylene glycol (PEG) 6000) and salinity (150 mM NaCl) stress. Proline contents in young seedlings greatly increased under both conditions after 3 h of treatment, whereas the levels of antioxidant enzymes remained unchanged. We assembled transcripts from the leaves and roots of rapeseed and performed BLASTN searches against the rapeseed genome database for the first time. Gene ontology analysis indicated that DEGs involved in catalytic activity, metabolic process, and response to stimulus were highly enriched. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that differentially expressed genes (DEGs) from the categories metabolic pathways and biosynthesis of secondary metabolites were highly enriched. We determined that myeloblastosis (MYB), NAM/ATAF1-2/CUC2 (NAC), and APETALA2/ethylene-responsive element binding proteins (AP2-EREBP) transcription factors function as major switches that control downstream gene expression and that proline plays a role under short-term abiotic stress treatment due to increased expression of synthesis and decreased expression of degradation. Furthermore, many common genes function in the response to both types of stress in this rapeseed.

Regulation of endogenous phytohormones alters the fluoranthene content in Arabidopsis thaliana.

Phytohormones are crucial endogenous modulators that regulate and integrate plant growth and responses to various environmental pollutants, including the uptake of pollutants into the plant. However, possible links between endogenous phytohormone pathways and pollutant accumulation are unclear. Here we describe the fluoranthene uptake, plant growth, and superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione S-transferase (GST) activities in relation to different endogenous phytohormones and different levels in Arabidopsis thaliana. Three phytohormone inhibitors-N-1-naphthyl-phthalamic acid (NPA), daminozide (DZ), and silver nitrate (SN)-were used to regulate endogenous auxin, gibberellin, and ethylene levels, respectively. Fluoranthene inhibited plant growth and root proliferation while increasing GST and SOD activity. The three inhibitors reduced fluoranthene levels in Arabidopsis by either affecting plant growth or modulating antioxidant enzyme activity. NPA reduced plant growth and increased CAT activity. SN promoted plant growth and increased POD and CAT activity, whereas DZ increased POD activity.

Characterization of Fructan Metabolism During Jerusalem Artichoke (Helianthus tuberosus L.) Germination.

The inulin-type fructans in Jerusalem artichoke (Helianthus tuberosus L.) tubers exhibit different degrees of polymerization and are critical for germination. We aimed to characterize the sugar metabolism dynamics in the tubers without bud eyes or shoots (T) and BE/S of indoor- and field-grown Jerusalem artichokes during germination. Ht1-FEH II and Ht1-FEH III (1-fructan exohydrolases II and III, inulin-degrading enzymes) expression increased 5 days after planting indoors, whereas Ht1-FEH II expression increased 72 days after planting in the field in T and BE/S. Ht1-SST (sucrose:sucrose 1-fructosyl transferase, inulin synthesis initiator), and Ht1-FFT (fructan:fructan 1-fructosyl transferase, inulin elongator) expression generally decreased in indoor-grown T. The enzyme activities of 1-FEH and 1-FFT were unchanged during germination in both indoor- and field-grown T and BE/S, whereas 1-SST activity decreased in indoor-grown T, while 1-FEH and 1-FFT activities increased as a function of germination time in BE/S of both indoor- and field-grown tubers. The total soluble sugar content gradually decreased in T after germination indoors or in the field, while at the end of germination, the sucrose and fructan contents decreased, and fructose content increased in the field. The enzyme activities of soluble vacuolar (VI) or neutral invertase (NI) did not change significantly, except at the late germination stage. Sucrose synthase (SS) and sucrose-phosphate synthase (SPS) activities were not significantly changed in T and BE/S in indoor-grown artichokes, while SS activity gradually increased, and SPS activity gradually decreased in field-grown artichokes, alongside sucrose degradation. Compared to T, BE/S generally had higher enzyme activities of 1-FEH and 1-FFT, promoting inulin hydrolysis. This work shows that the process of tuber germination is similar indoors and in the field, and germination studies can therefore be conducted in either environment.

Expression and purification of plant fructan exohydrolases and their potential applications in fructose production.

Inulinases from microorganisms have been extensively studied for their role in the production of fructose from fructan. Fructan can also be hydrolyzed by plant fructan exohydrolases (FEHs), but these enzymes have not been used to produce fructose commercially. Two Ht1-FEHs (Ht1-FEH I and Ht1-FEH II) were recently characterized in Jerusalem artichoke. In this study, we cloned the third member of the Ht1-FEH family in Jerusalem artichoke (i.e., Ht1-FEH III). When heterologously expressed in Pichia pastoris X-33, Ht1-FEH III not only demonstrated hydrolysis activity towards ß (2, 1)-linked fructans and ß (2, 6)-linked levan, but also towards sucrose. To explore the potential industrial applications, we heterologously expressed and purified six plant 1-FEHs from two typical fructan plants (i.e., chicory and Jerusalem artichoke) and showed that chicory Ci1-FEH IIa had the highest hydrolysis capacity to fructan in vitro. Furthermore, we immobilized Ci1-FEH IIa on resin and optimized the immobilization conditions. We found that inulin-type fructan or the tuber extract from Jerusalem artichoke could be rapidly degraded into fructose and sucrose by immobilized Ci1-FEH IIa. The capacity of Ci1-FEH IIa to release fructose from fructans was comparable to that of some inulinases from microorganisms. Thus, plant FEHs have potential applications in fructose production.

Characterization of the biosorption and biodegradation properties of Ensifer adhaerens: A potential agent to remove polychlorinated biphenyls from contaminated water.

Ensifer adhaerens is a soil bacterium known for its potential to remove pollutants from the environment. We investigated the contributions of biosorption and biodegradation to the process of polychlorinated biphenyl (PCB) removal from water by living or heat-killed E. adhaerens with different incubation times. We examined the physicochemical properties of E. adhaerens, including its membrane surface moieties, extracellular polymeric substances, and defense-related enzyme activities. In addition, we measured the biosorption and biodegradation of different PCB congeners. We found that removal of PCBs by heat-killed E. adhaerens was attributed to biosorption only, while both biosorption and biodegradation were responsible for the dissipation of PCBs by live E. adhaerens. Biosorption initially plays a major role in PCB removal, but biodegradation becomes increasingly important with increased incubation time. The results of infrared spectroscopy analysis showed that bacterial lipids, proteins, and polysaccharides, which offer abundant binding sites, are responsible for the biosorption of PCBs. Biodegradation was correlated with loosely bound polysaccharides and defense-related enzyme activities that could increase the pollutant's solubility and facilitate further degradation. The PCB congeners exhibited different biosorption and biodegradation patterns, and the patterns were correlated with the octanol-water partition coefficients (Kow) of the congeners. The more hydrophobic organic compounds tended to have higher biosorption, but lower biodegradation capacities. These results indicate that E. adhaerens-mediated biosorption and biodegradation of PCBs are dependent on the status of the strain, the incubation time, and the PCB congener present, and suggest guidelines for PCB removal from water.

Cloning and functional characterization of two abiotic stress-responsive Jerusalem artichoke (Helianthus tuberosus) fructan 1-exohydrolases (1-FEHs).

Two fructan hydrolases were previously reported to exist in Jerusalem artichoke (Helianthus tuberosus) and one native fructan-ß-fructosidase (1-FEH) was purified to homogeneity by SDS-PAGE, but no corresponding cDNA was cloned. Here, we cloned two full-length 1-FEH cDNA sequences from Jerusalem artichoke, named Ht1-FEH I and Ht1-FEH II, which showed high levels of identity with chicory 1-FEH I and 1-FEH II. Functional characterization of the corresponding recombinant proteins in Pichia pastoris X-33 demonstrated that both Ht1-FEHs had high levels of hydrolase activity towards ß(2,1)-linked fructans, but low or no activity towards ß(2,6)-linked levan and sucrose. Like other plant FEHs, the activities of the recombinant Ht1-FEHs were greatly inhibited by sucrose. Real-time quantitative PCR analysis showed that Ht1-FEH I transcripts accumulated to high levels in the developing leaves and stems of artichoke, whereas the expression levels of Ht1-FEH II increased in tubers during tuber sprouting, which implies that the two Ht1-FEHs play different roles. The levels of both Ht1-FEH I and II transcript were significantly increased in the stems of NaCl-treated plants. NaCl treatment also induced transcription of both Ht1-FEHs in the tubers, while PEG treatments slightly inhibited the expression of Ht1-FEH II in tubers. Analysis of sugar-metabolizing enzyme activities and carbohydrate concentration via HPLC showed that the enzyme activities of 1-FEHs were increased but the fructose content was decreased under NaCl and PEG treatments. Given that FEH hydrolyzes fructan to yield Fru, we discuss possible explanations for the inconsistency between 1-FEH activity and fructan dynamics in artichokes subjected to abiotic stress.

Multiple NUCLEAR FACTOR Y transcription factors respond to abiotic stress in Brassica napus L.

Members of the plant NUCLEAR FACTOR Y (NF-Y) family are composed of the NF-YA, NF-YB, and NF-YC subunits. In Brassica napus (canola), each of these subunits forms a multimember subfamily. Plant NF-Ys were reported to be involved in several abiotic stresses. In this study, we demonstrated that multiple members of thirty three BnNF-Ys responded rapidly to salinity, drought, or ABA treatments. Transcripts of five BnNF-YAs, seven BnNF-YBs, and two BnNF-YCs were up-regulated by salinity stress, whereas the expression of thirteen BnNF-YAs, ten BnNF-YBs, and four BnNF-YCs were induced by drought stress. Under NaCl treatments, the expression of one BnNF-YA10 and four NF-YBs (BnNF-YB3, BnNF-YB7, BnNF-YB10, and BnNF-YB14) were greatly increased. Under PEG treatments, the expression levels of four NF-YAs (BnNF-YA9, BnNF-YA10, BnNF-YA11, and BnNF-YA12) and five NF-YBs (BnNF-YB1, BnNF-YB8, BnNF-YB10, BnNF-YB13, and BnNF-YB14) were greatly induced. The expression profiles of 20 of the 27 salinity- or drought-induced BnNF-Ys were also affected by ABA treatment. The expression levels of six NF-YAs (BnNF-YA1, BnNF-YA7, BnNF-YA8, BnNF-YA9, BnNF-YA10, and BnNF-YA12) and seven BnNF-YB members (BnNF-YB2, BnNF-YB3, BnNF-YB7, BnNF-YB10, BnNF-YB11, BnNF-YB13, and BnNF-YB14) and two NF-YC members (BnNF-YC2 and BnNF-YC3) were greatly up-regulated by ABA treatments. Only a few BnNF-Ys were inhibited by the above three treatments. Several NF-Y subfamily members exhibited collinear expression patterns. The promoters of all stress-responsive BnNF-Ys harbored at least two types of stress-related cis-elements, such as ABRE, DRE, MYB, or MYC. The cis-element organization of BnNF-Ys was similar to that of Arabidopsis thaliana, and the promoter regions exhibited higher levels of nucleotide sequence identity with Brassica rapa than with Brassica oleracea. This work represents an entry point for investigating the roles of canola NF-Y proteins during abiotic stress responses and provides insight into the genetic evolution of Brassica NF-Ys.

Identification and characterization of NF-Y transcription factor families in Canola (Brassica napus L.).

NF-Y (NUCLEAR FACTOR-Y), a heterotrimeric transcription factor, is composed of NF-YA, NF-YB, and NF-YC proteins in yeast, animal, and plant systems. In plants, each of the NF-YA/B/C subunit forms a multi-member family. NF-Ys are key regulators with important roles in many physiological processes, such as drought tolerance, flowering time, and seed development. In this study, we identified, annotated, and further characterized 14 NF-YA, 14 NF-YB, and 5 NF-YC proteins in Brassica napus (canola). Phylogenetic analysis revealed that the NF-YA/B/C subunits were more closely clustered with the Arabidopsis thaliana (Arabidopsis) homologs than with rice OsHAP2/3/5 subunits. Analyses of the conserved domain indicated that the BnNF-YA/B/C subfamilies, respectively, shared the same conserved domains with those in other organisms, including Homo sapiens, Saccharomyces cerevisiae, Arabidopsis, and Oryza sativa (rice). An examination of exon/intron structures revealed that most gene structures of BnNF-Y were similar to their homologs in Arabidopsis, a model dicot plant, but different from those in the model monocot plant rice, suggesting that plant NF-Ys diverged before monocot and dicot plants differentiated. Spatial-tempo expression patterns, as determined by qRT-PCR, showed that most BnNF-Ys were widely expressed in different tissues throughout the canola life cycle and that several closely related BnNF-Y subunits had similar expression profiles. Based on these findings, we predict that BnNF-Y proteins have functions that are conserved in the homologous proteins in other plants. This study provides the first extensive evaluation of the BnNF-Y family, and provides a useful foundation for dissecting the functions of BnNF-Y.

Salt stress encourages proline accumulation by regulating proline biosynthesis and degradation in Jerusalem artichoke plantlets.

Proline accumulation is an important mechanism for osmotic regulation under salt stress. In this study, we evaluated proline accumulation profiles in roots, stems and leaves of Jerusalem artichoke (Helianthus tuberosus L.) plantlets under NaCl stress. We also examined HtP5CS, HtOAT and HtPDH enzyme activities and gene expression patterns of putative HtP5CS1, HtP5CS2, HtOAT, HtPDH1, and HtPDH2 genes. The objective of our study was to characterize the proline regulation mechanisms of Jerusalem artichoke, a moderately salt tolerant species, under NaCl stress. Jerusalem artichoke plantlets were observed to accumulate proline in roots, stems and leaves during salt stress. HtP5CS enzyme activities were increased under NaCl stress, while HtOAT and HtPDH activities generally repressed. Transcript levels of HtP5CS2 increased while transcript levels of HtOAT, HtPDH1 and HtPDH2 generally decreased in response to NaCl stress. Our results supports that for Jerusalem artichoke, proline synthesis under salt stress is mainly through the Glu pathway, and HtP5CS2 is predominant in this process while HtOAT plays a less important role. Both HtPDH genes may function in proline degradation.

Expression and functional analysis of NUCLEAR FACTOR-Y, subunit B genes in barley.

NUCLEAR FACTOR-Y, subunit B (NF-YB) comprises a multigene family in plants and has been shown to play important roles in growth, development, and response to environmental stress. In this study, five NF-YBs containing the full-length coding region were obtained from barley (Hordeum vulgare) through database sequence analysis, cloning, and sequencing. Sequence alignment and phylogenetic analysis showed that HvNF-YB3 and HvNF-YB1 were clustered with NF-YB2 and NF-YB3 in Arabidopsis, suggesting these NF-YBs are evolutionary and functionally related. To test this hypothesis, HvNF-YB3 and HvNF-YB1 were overexpressed in Arabidopsis. Overexpression of HvNF-YB1 greatly promoted early flowering in Arabidopsis, supporting that HvNF-YB1may have conserved gene function in flowering time control as NF-YB2 and NF-YB3 in Arabidopsis. Overexpression of HvNF-YB3 in Arabidopsis had no effect on flowering time. An analysis of barley single-nucleotide polymorphism (SNP) data, however, revealed a significant association between an HvNF-YB3 SNP and heading date. While it is unknown whether HvNF-YB3 directly contributes to heading date regulation, the results implied that HvNF-YB3 may also have conserved function in flowering time (heading date in barley) control. Further studies are needed to directly verify these gene functions in barley. Barley NF-YBs showed different expression patterns associated with tissue types, developmental stages, and response to different stress treatments, suggesting that barley NF-YBs may be involved in other physiological processes.

A putative CCAAT-binding transcription factor is a regulator of flowering timing in Arabidopsis.

Flowering at the appropriate time of year is essential for successful reproduction in plants. We found that HAP3b in Arabidopsis (Arabidopsis thaliana), a putative CCAAT-binding transcription factor gene, is involved in controlling flowering time. Overexpression of HAP3b promotes early flowering while hap3b, a null mutant of HAP3b, is delayed in flowering under a long-day photoperiod. Under short-day conditions, however, hap3b did not show a delayed flowering compared to wild type based on the leaf number, suggesting that HAP3b may normally be involved in the photoperiod-regulated flowering pathway. Mutant hap3b plants showed earlier flowering upon gibberellic acid or vernalization treatment, which means that HAP3b is not involved in flowering promoted by gibberellin or vernalization. Further transcript profiling and gene expression analysis suggests that HAP3b can promote flowering by enhancing expression of key flowering time genes such as FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Our results provide strong evidence supporting a role of HAP3b in regulating flowering in plants grown under long-day conditions.

Expression of a putative laccase gene, ZmLAC1, in maize primary roots under stress.

Laccases are multi-copper-containing glycoproteins and comprise a multi-gene family in plants. However, their physiological functions are still not well understood. We obtained sequence information for a putative laccase gene, ZmLAC1, from maize and studied ZmLAC1 expression in detail. The deduced ZmLAC1 protein was 70% identical to LpLAC5-4, a laccase from ryegrass. ZmLAC1 was expressed in leaves, stems and roots of maize seedlings. In unstressed maize primary roots, a higher ZmLAC1 transcript level was located in the basal region where cell elongation had ceased compared to the apical 5 mm of the roots where cells were rapidly dividing and elongating. A treatment with 300 mM NaCl resulted in a shortened root elongation zone (< 2 mm) and swelling in the apical 5 mm. Associated with the morphological change, the transcript level of ZmLACl was enhanced in the apical 5 mm, reaching a level similar to that in the basal region. Other abiotic stresses tested--such as 28.5% polyethylene glycol (PEG), which caused an inhibition of root elongation comparable to 300 mM NaCl--did not affect ZmLAC1 transcript level. Potential roles of ZmLAC1 in the roots responding to NaCl or other high concentration of salts are discussed.

Mutant identification and characterization of the laccase gene family in Arabidopsis.

Laccases, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductases, are multi-copper containing glycoproteins. Despite many years of research, genetic evidence for the roles of laccases in plants is mostly lacking. In this study, a reverse genetics approach was taken to identify T-DNA insertional mutants (the SALK collection) available for genes in the Arabidopsis laccase family. Twenty true null mutants were confirmed for 12 laccase genes of the 17 total laccase genes (AtLAC1 to AtLAC17) in the family. By examining the mutants identified, it was found that four mutants, representing mutations in three laccase genes, showed altered phenotypes. Mutants for AtLAC2, lac2, showed compromised root elongation under PEG-induced dehydration conditions; lac8 flowered earlier than wild-type plants, and lac15 showed an altered seed colour. The diverse phenotypes suggest that laccases perform different functions in plants and are not as genetically redundant as previously thought. These mutants will prove to be valuable resources for understanding laccase functions in vivo.

Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis.

Laccase, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductase, has been proposed to be involved in lignin synthesis in plants based on its in vitro enzymatic activity and a close correlation with the lignification process in plants. Despite many years of research, genetic evidence for the role of laccase in lignin synthesis is still missing. By screening mutants available for the annotated laccase gene family in Arabidopsis, we identified two mutants for a single laccase gene, AtLAC15 (At5g48100) with a pale brown or yellow seed coat which resembled the transparent testa (tt) mutant phenotype. A chemical component analysis revealed that the mutant seeds had nearly a 30% decrease in extractable lignin content and a 59% increase in soluble proanthocyanidin or condensed tannin compared with wild-type seeds. In an in vitro enzyme assay, the developing mutant seeds showed a significant reduction in polymerization activity of coniferyl alcohol in the absence of H(2)O(2). Among the dimers formed in the in vitro assay using developing wild-type seeds, 23% of the linkages were beta-O-4 which resembles the major linkages formed in native lignin. The evidence strongly supports that AtLAC15 is involved in lignin synthesis in plants. To our knowledge, this is the first genetic evidence for the role of laccase in lignin synthesis. Changes in seed coat permeability, seed germination and root elongation were also observed in the mutant.