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Liang M.,Nanjing Agricultural University | Liang M.,Jiangsu Key Laboratory of Marine Biology | Yin X.,Nanjing Agricultural University | Yin X.,Jiangsu Key Laboratory of Marine Biology | And 7 more authors.
Planta | Year: 2014

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. © 2013 Springer-Verlag Berlin Heidelberg.

Zhang X.,Nanjing Agricultural University | Zhang X.,Jiangsu Key Laboratory of Marine Biology | Xu H.,Nanjing Agricultural University | Xu H.,Jiangsu Key Laboratory of Marine Biology | And 8 more authors.
Bioenergy Research | Year: 2016

Bioethanol is a promising renewable source of energy. The heterologous expression of inulinases from microorganisms in the yeast Saccharomyces cerevisiae improves ethanol production from inulin. Fructan exohydrolases (FEHs) from fructan-rich plants hydrolyze fructofuranosyl units in inulin to produce fructose. Here, we examined whether the heterologous expression of FEHs could also improve ethanol production in yeast. First, we expressed two Jerusalem artichoke (Helianthus tuberosus) FEH genes (Ht1-FEH I and II) in Pichia pastoris yeast X-33 to examine the biochemical properties of the encoded enzymes. Ht1-FEH I was relatively stable at pH 4–8 and 4–35 °C and Ht1-FEH II was relatively stable at pH 4–8 and 4–40 °C. The Km and Vm values of Ht1-FEH I were 0.68 and 0.00129 mg/min, while those of Ht1-FEH II were 0.92 and 0.0048 mg/min, respectively. The enzyme activities were affected by metal ions and protein inhibitors. Additionally, the transgenic expression of Ht1-FEH I and Ht1-FEH II in S. cerevisiae 6525 at pH 6, 30 °C resulted in 25 and 27 % increases in ethanol production compared to the non-FEH-transformed control (CK), respectively. The efficiency of ethanol production was greater in yeast expressing plant FEHs than in yeast expressing inulinases derived from some microorganisms. Thus, plant FEHs have potential applications in bioethanol production. © 2016 Springer Science+Business Media New York

Xu H.,Nanjing Agricultural University | Xu H.,Jiangsu Key Laboratory of Marine Biology | Liang M.,Nanjing Agricultural University | Liang M.,Jiangsu Key Laboratory of Marine Biology | And 9 more authors.
Plant Molecular Biology | Year: 2014

Two fructan hydrolases were previously reported to exist in Jerusalem artichoke (Helianthus tuberosus) and one native fructan-β-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 β(2,1)-linked fructans, but low or no activity towards β(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. © 2014, Springer Science+Business Media Dordrecht.

Liang M.,Nanjing Agricultural University | Liang M.,Jiangsu Key Laboratory of Marine Biology | Lin M.,Nanjing Agricultural University | Lin M.,Jiangsu Key Laboratory of Marine Biology | And 9 more authors.
Plant Growth Regulation | Year: 2015

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. © 2014, Springer Science+Business Media Dordrecht.

Xu L.,Nanjing Agricultural University | Lin Z.,Nanjing Agricultural University | Lin Z.,Jiangsu Key Laboratory of Marine Biology | Tao Q.,Nanjing Agricultural University | And 9 more authors.
PLoS ONE | Year: 2014

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 BnNFYB14) were greatly increased. Under PEG treatments, the expression levels of four NF-YAs (BnNF-YA9, BnNF-YA10, BnNFYA11, 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. Copyright © 2014 Xu et al.

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