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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 β (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.

The present study was carried out to assess the underlying physiological and biochemical events of salinity adaptation in blessed thistle (Silybum marianum Gaertn.) and peppermint (Mentha haplocalyx Briq.). The seedlings of both species were exposed to four different salt (NaCl) concentrations: 30 mM (3.8 mS cm–1), 60 mM (7 mS cm–1), 120 mM (13.4 mS cm–1), and 180 mM (19.6 mS cm–1), and the changes in biomass, osmoprotectant accumulation, ion concentrations, and antioxidant enzymes activity were recorded every 15 days in blessed thistle and every 10 days in peppermint for three samplings. The results showed that salinity significantly increased malondialdehyde (MDA) and proline accumulation in leaves and roots (3- to fourfold) but had little effect on the chlorophyll content of leaves at the first sampling in both species. The soluble protein content of blessed thistle significantly increased after salt treatment at the first sampling (83% in roots and 55% in the aerial parts of plants), whereas peppermint was less affected. K+/Na+ and Ca2+/Na+ ratios in both species exposed to 180 mM NaCl were more than tenfold lower than the untreated control at all three samplings. Superoxide dismutase (SOD) and catalase (CAT) activity were significantly affected by salinity in blessed thistle but not in peppermint, while peroxidase (POD) activity increased with increasing salinity in peppermint but not in blessed thistle at all three samplings. These results suggested blessed thistle and peppermint utilized different tolerance strategies which shedding new light on how to grow halophytes in saline lands.

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.

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 β (2, 1)-linked fructans and β (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.

In this study, we examined whether vermicompost enhances a plant’s tolerance to salinity. We analyzed the physiological responses of the aerial parts and roots of the herbal stress-resistant plants blessed thistle and peppermint with NaCl and cow manure vermicompost and inorganic fertilizer. Salinity greatly enhanced the accumulation of malondialdehyde (MDA) and proline in the aerial parts and roots of the two species, but did not affect chlorophyll content. The K + /Na + and Ca 2+ /Na + ratios and the total soluble protein content were decreased in the aerial parts and roots under salinity conditions in both species. Under normal conditions, vermicompost enhanced plant growth and increased the K + /Na + and Ca 2+ /Na + ratios and total soluble protein content while inorganic fertilizer treatment increased the total soluble protein content and proline content in both species. Proline content in both species was greatly decreased under vermicompost treatment than inorganic treatment under normal conditions. Furthermore, under high salinity conditions, vermicompost treatment significantly reduced the MDA content and increased total soluble protein content and the K + /Na + and Ca 2+ /Na + ratios than stress-treated plants. Vermicompost had complex effects on the antioxidant enzyme activities of plants grown under high salinity treatment. Our results show vermicompost mitigates the effects of 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.

The NF-Y (NUCLEAR FACTOR-Y) transcription factor in plants is composed of NF-YA, NF-YB, and NF-YC subunits. Each of the NF-YA/B/C subunit types is represented by a large family of proteins that functions in growth, stress regulation, and other processes. In a previous study, we identified 14 NF-YAs, 14 NF-YBs, and 5 NF-YCs in Brassica napus (rapeseed) using an expressed sequence tag (EST) database. Building on this earlier work, we identified and characterized an additional 9 NF-YAs, 6 NF-YBs, and 5 NF-YCs using the completed rapeseed genome sequence in the CoGe database. Conserved domain, phylogenetic, and exon–intron structure analysis revealed that the newly identified subunits were closely clustered with the NF-Y subunits from Arabidopsis thaliana and the previously identified NF-Y subunits from rapeseed. Expression analysis under abscisic acid (ABA) treatment, salinity stress, and drought stress, as determined by qRT-PCR, showed that all of the newly identified BnNF-Y subunits were responsive to at least one type of abiotic stress. Several closely related BnNF-Y subunits had similar conserved domains, gene structures, and transcriptional profiles. Under drought, salinity, and ABA treatments, Arabidopsis plants heterologously expressing BnNF-YA3 had delayed seed germination, slower root elongation, shorter total root lengths, and fewer root tips than untransformed Arabidopsis, suggesting that BnNF-YA3 functions in the abiotic stress response. Thus, we have identified over 50 BnNF-Y subunit genes in rapeseed to date, and demonstrated that BnNF-YA3 takes part in the abiotic stress response.

Na+/H+ antiporters (NHXs) primarily catalyze the exchange of Na+ for H+ across vacuole membranes. A novel vacuolar Na+/H+ exchanger, CiNHX1, was cloned from chicory (Cichorium intybus L.), which contains an open reading frame of 1,644 bp. Sequence alignment and phylogenetic analysis indicated that CiNHX shared a great degree of similarity with reported class-I NHX sequences within predicted transmembrane segments and an amiloride-binding domain. Quantitative real-time PCR analysis revealed that salt stress, unlike abscisic acid (ABA) or osmotic stress, greatly induced the expression of CiNHX1, suggesting that CiNHX1 is mainly involved in ABA-independent stress signaling pathways. The fact that chicory accumulated more Na+ compared to untreated plants under salt stress was concordant to the higher levels of CiNHX mRNA under salinity. A heterologous expression of CiNHX1 in Saccharomyces cerevisiae mutant suggested that CiNHX1 could mimic the function of the endogenous NHX1 protein. Subcellular localization assay revealed that CiNHX1 was a tonoplast membrane-localized protein. These results suggested that CiNHX1 plays a critical role in chicory’s tolerance to salinity stress.

Two novel DREB (dehydration-responsive element-binding protein) genes, designated as CiDREB1A and CiDREB1B, were cloned from chicory (Cichorium intybus). Both of these genes contained a conserved EREBP/AP2 domain and were classified into the A-1 subgroup of the DREB subfamily based on phylogenetic analysis. Quantitative real-time PCR analysis revealed that low temperature, but not ABA, greatly induced the expression of both CiDREB1 genes, suggesting that these genes are involved in ABA-independent stress signaling pathways. A subcellular localization assay revealed that both CiDREBs localized to the nucleus. In addition, we showed by yeast one-hybrid analysis that these two CiDREB proteins bind to the DRE motif of RD19A. These results suggest that CiDREB1A and CiDREB1B are important regulators of stress-responsive signaling in chicory.

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.

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.

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.

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.

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.

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.

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.