Binder Jetting Additive Manufacturing of High Porosity 316L Stainless Steel Metal Foams.

High porosity (40% to 60%) 316L stainless steel containing well-interconnected open-cell porous structures with pore openness index of 0.87 to 1 were successfully fabricated by binder jetting and subsequent sintering processes coupled with a powder space holder technique. Mono-sized (30 µm) and 30% (by volume) spherically shaped poly(methyl methacrylate) (PMMA) powder was used as the space holder material. The effects of processing conditions such as: (1) binder saturation rates (55%, 100% and 150%), and (2) isothermal sintering temperatures (1000 ○C to 1200 ○C) on the porosity of 316L stainless steel parts were studied. By varying the processing conditions, porosity of 40% to 45% were achieved. To further increase the porosity values of 316L stainless steel parts, 30 vol. % (or 6 wt. %) of PMMA space holder particles were added to the 3D printing feedstock and porosity values of 57% to 61% were achieved. Mercury porosimetry results indicated pore sizes less than 40 µm for all the binder jetting processed 316L stainless steel parts. Anisotropy in linear shrinkage after the sintering process was observed for the SS316L parts with the largest linear shrinkage in the Z direction. The Young's modulus and compression properties of 316L stainless steel parts decreased with increasing porosity and low Young's modulus values in the range of 2 GPa to 29 GPa were able to be achieved. The parts fabricated by using pure 316L stainless steel feedstock sintered at 1200 ○C with porosity of ~40% exhibited the maximum overall compressive properties with 0.2% compressive yield strength of 52.7 MPa, ultimate compressive strength of 520 MPa, fracture strain of 36.4%, and energy absorption of 116.7 MJ/m3, respectively. The Young's modulus and compression properties of the binder jetting processed 316L stainless steel parts were found to be on par with that of the conventionally processed porous 316L stainless steel parts and even surpassed those having similar porosities, and matched to that of the cancellous bone types.

Characterization of a glutamine synthetase gene DvGS1 from Dunaliella viridis and investigation of the impact on expression of DvGS1 in transgenic Arabidopsis thaliana.

A novel glutamine synthetase (GS) gene DvGS1 showing highest amino acid sequence identity of 78 % with the other homologous GS proteins from green algae, was isolated and characterized from Dunaliella viridis. Phylogenetic analysis revealed that DvGS1 occupied an independent phylogenetic position which was different with the GSs from higher plants, animals and microbes. Functional complement in E. coli mutant confirmed that the DvGS1 encoded functional GS enzyme. Real-time PCR analysis of DvGS1 in D. viridis cells under nitrogen starvation revealed that the mRNA level of DvGS1 was positively up-regulated in 12 h. The DvGS1 levels at the points of 12 and 24 h were separately twofold and fourfold of the level before nitrogen starvation. In order to investigate the potential application of DvGS1 in higher plants, the transgenic study of DvGS1 in Arabidopsis thaliana was carried out. Phenotype identification demonstrated that all three transgenic lines of T3 generation showed obviously enhanced root length (26 %), fresh weight (22-46 % at two concentrations of nitrate supplies), stem length (26 %), leaf size (29 %) and silique number (30 %) compared with the wild-type Arabidopsis. Biochemical analysis confirmed that all three transgenic lines had higher total nitrogen content, soluble protein concentration, total amino acid content and the leaf GS activity than the wild type plants. The free NH4 (+) and NO3 (-) concentration in fresh leaves of three transgenic lines were reduced by 17-26 % and 14-15 % separately (at two concentrations of nitrate supplies) compared with those of the wild types. All the results indicated that over-expression of DvGS1 in Arabidopsis significantly results in the improvement of growth phenotype and the host's nitrogen use efficiency.

Characterization of a glutamine synthetase gene DvGS2 from Dunaliella viridis and biochemical identification of DvGS2-transgenic Arabidopsis thaliana.

The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella be a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%-48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%-43%), total GS activity (39%-45%) and soluble protein concentration (23%-24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency.

Characterization of an Ac transposon system based on apt1-m1 (Ac) on the long arm of maize chromosome 9.

Activator/Dissociation (Ac/Ds) transposable elements have been used in maize insertional mutagenesis as a complement to Mutator (Mu). In this study, to further improve the efficiency of the Ac/Ds mutagenesis system, we adopted apt1-m1 (Ac) on the long arm of chromosome 9 (9L) as a donor Ac to create an Ac insertion library. This system is based on the negative selection pressure against the donor Ac, and it was highly efficient for isolating new transposition events. We obtained 9,625 transposition events from 1083 F1 ears with an average transposition rate of 8.66 % (rates ranged from 1.11 to 29.73 %). We also adopted a modified PCR-based genome walking strategy to improve the efficiency of the new method for isolating transposon-flanking sequences. This method is more efficient than the Southern-based method that was used in previous studies. A validation step was developed to distinguish transposon tags derived from newly transposed Ac or Ds elements. Using this PCR-based method, we isolated 67 inheritable flanking sequences from the apt1-m1 (Ac) transposition library; of these, 51 were confirmed as tr-Ac-flanking sequences and 11 were tr-Ds-flanking sequences. Similar to other Ac donors from different loci, the apt1-m1 (Ac) system also exhibited a preference for short distance transposition. In this study, we have further improved the Ac mutagenesis system in maize for gene isolation and functional genomics studies.

Zea mays Taxilin protein negatively regulates opaque-2 transcriptional activity by causing a change in its sub-cellular distribution.

Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.

Opaque1 encodes a myosin XI motor protein that is required for endoplasmic reticulum motility and protein body formation in maize endosperm.

Myosins are encoded by multigene families and are involved in many basic biological processes. However, their functions in plants remain poorly understood. Here, we report the functional characterization of maize (Zea mays) opaque1 (o1), which encodes a myosin XI protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. Compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (ER) structures and an increased number of smaller, misshapen protein bodies. The O1 gene was isolated by map-based cloning and was shown to encode a member of the plant myosin XI family (myosin XI-I). In endosperm cells, the O1 protein is associated with rough ER and protein bodies. Overexpression of the O1 tail domain (the C-terminal 644 amino acids) significantly inhibited ER streaming in tobacco (Nicotiana benthamiana) cells. Yeast two-hybrid analysis suggested an association between O1 and the ER through a heat shock protein 70-interacting protein. In summary, this study indicated that O1 influences protein body biogenesis by affecting ER morphology and motility, ultimately affecting endosperm texture.

Molecular characterization of a genomic interval with highly uneven recombination distribution on maize chromosome 10 L.

Homologous recombination in meiosis provides the evolutionary driving force in eukaryotic organisms by generating genetic variability. Meiotic recombination does not always occur evenly across the chromosome, and therefore genetic and physical distances are not consistently in proportion. We discovered a 278 kb interval on the long arm of chromosome 10 (10 L) through analyzed 13,933 descendants of backcross population. The recombinant events distributed unevenly in the interval. The ratio of genetic to physical distance in the interval fluctuated about 47-fold. With the assistance of molecular markers, the interval was divided into several subintervals for further characterization. In agreement with previous observations, high gene-density regions such as subinterval A and B were also genetic recombination hot subintervals, and repetitive sequence-riched region such as subinterval C was also found to be recombination inert at the detection level of the study. However, we found an unusual subinterval D, in which the 72-kb region contained 6 genes. The gene-density of subinterval D was 5.8 times that of the genome-wide average. The ratio of genetic to physical distance in subinterval D was 0.58 cM/Mb, only about 3/4 of the genome average. We carried out an analysis of sequence polymorphisms and methylation status in subinterval D, and the potential causes of recombination suppression were discussed. This study was another case of a detailed genetic analysis of an unusual recombination region in the maize genome.

Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm.

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.

The characterization of two peroxiredoxin genes in Dunaliella viridis provides insights into antioxidative response to salt stress.

Peroxiredoxins (Prxs), a group of antioxidant enzymes, are an important component of the oxidative defense system and have been demonstrated to function as peroxidases, sensors of H(2)O(2)-mediated signaling and/or chaperones. In this study, a cDNA library was constructed from a halotolerant alga, Dunaliella viridis, and was used in a functional complementation screen for antioxidative genes in an oxidative sensitive yeast mutant. Two Prx genes, DvPrx1 and DvPrx2, were obtained from this screen. These two genes were classified as type II Prx and 2-Cys Prx based on amino acid sequence and phylogenetic analysis. When over-expressed in yeast cells, both Prx genes were able to confer better oxidative tolerance and decrease the level of reactive oxygen species (ROS). Subcellular localization experiments in tobacco cells revealed that both DvPrx1 and DvPrx2 were localized in the cytosol. The transcription of DvPrx1 and DvPrx2 can be induced by hypersalinity shock, but is not obviously affected by treatment with high levels of oxidant. Our results shed light on the function and regulation of Prx genes from Dunaliella and their potential roles in salt tolerance.

Molecular cloning and characterization of a trehalose-6-phosphate synthase/phosphatase from Dunaliella viridis.

Dunaliella is a group of green algae with exceptional stress tolerance capability, and is considered as an important model organism for stress tolerance study. Here we cloned a TPS (trehalose-6-phosphate synthase) gene from Dunaliella viridis and designated it as DvTPS (D. viridis trehalose-6-phosphate synthase/phosphatase).The DvTPS cDNA contained an ORF of 2793 bp encoding 930 aa. DvTPS had both TPS and TPP domain and belonged to the Group II TPS/TPP fusion gene family. Southern blots showed it has a single copy in the genome. Genome sequence analysis revealed that it has 18 exons and 17 introns. DvTPS had a constitutive high expression level under various NaCl culture conditions, however, could be induced by salt shock. Promoter analysis indicated there were ten STREs (stress response element) in its promoter region, giving a possible explanation of its inducible expression pattern upon salt shock. Yeast functional complementation analysis showed that DvTPS had neither TPS nor TPP activity. However, DvTPS could improve the salt tolerance of yeast salt sensitive mutant G19. Our results indicated that despite DvTPS showed significant similarity with TPS/TPP, its real biological function is still remained to be revealed.

Molecular cloning and characterization of a vacuolar H+₋pyrophosphatase from Dunaliella viridis.

The halotolerant alga Dunaliella adapts to exceptionally high salinity and possesses efficient mechanisms for regulating intracellular Na(+). In plants, sequestration of Na(+) into the vacuole is driven by the electrochemical H(+) gradient generated by H(+) pumps, and this Na(+) sequestration is one mechanism that confers salt tolerance to plants. To investigate the role of vacuolar H(+) pumps in the salt tolerance of Dunaliella, we isolated the cDNA of the vacuolar proton-translocating inorganic pyrophosphatase (V-H(+)-PPase) from Dunaliella viridis. The DvVP cDNA is 2,984 bp in length, codes for a polypeptide of 762 amino acids and has 15 transmembrane domains. The DvVP protein is highly similar to V-H(+)-PPases from other green algae and higher plant species, in terms of its amino acid sequence and its transmembrane model. A phylogenetic analysis of V-H(+)-PPases revealed the close relationship of Dunaliella to green algal species of Charophyceae and land plants. The heterologous expression of DvVP in the yeast mutant G19 (Δena1-4) suppressed Na(+) hypersensitivity, and a GFP-fusion of DvVP localized to the vacuole membranes in yeast, indicating that DvVP encodes a functional V-H(+)-PPase. A northern blot analysis showed a decrease in the transcript abundance of DvVP at higher salinity in D. viridis cells, which is in contrast to the salt-induced upregulation of V-H(+)-PPase in some plants, suggesting that the expression of DvVP under salt stress may be regulated by different mechanisms in Dunaliella. This study not only enriched our knowledge about the biological functions of V-H(+)-PPases in different organisms but also improved our understanding of the molecular mechanism of salt tolerance in Dunaliella.

The cloning and characterization of two ammonium transporters in the salt-resistant green alga, Dunaliella viridis.

Ammonium (NH(4) (+)) transport is a key process in nitrogen metabolism. To elucidate the role of ammonium transporters in the nitrogen consumption of the salt-resistant green alga, Dunaliella viridis, two ammonium transporter genes, DvAMT1;1 and DvAMT1;2, were isolated from cDNA libraries of D. viridis. DvAMT1;1 and DvAMT1;2 share only 40% amino acid identity, indicating that they have highly divergent coding sequences. Functional complementation in a yeast mutant defective in ammonium uptake indicated that both DvAMT1;1 and DvAMT1;2 were functional ammonium transporters. Quantitative RT-PCR showed similar expression patterns, but different transcript abundance levels, for DvAMT1;1 and DvAMT1;2 under different nitrogen conditions. Both were induced at low nitrogen and inhibited at high nitrogen concentrations, especially when NH(4) (+) was the nitrogen source. At the transcriptional level, DvAMT1;1 was diurnally regulated, while DvAMT1;2 was not. In addition, under NaCl concentrations that ranged from 0.5 to 3 M, DvAMT1;1 was down-regulated at the higher salt conditions; conversely, DvAMT1;2 maintained a relatively low, but stable, transcript abundance. The observed differences in transcriptional regulation of DvAMT1;1 and DvAMT1;2 are indicative of their diverse physiological functions in D. viridis.

An induced hypersensitive-like response limits expression of foreign peptides via a recombinant TMV-based vector in a susceptible tobacco.

BACKGROUND: By using tobacco mosaic virus (TMV)-based vectors, foreign epitopes of the VP1 protein from food-and-month disease virus (FMDV) could be fused near to the C-terminus of the TMV coat protein (CP) and expressed at high levels in susceptible tobacco plants. Previously, we have shown that the recombinant TMV vaccines displaying FMDV VP1 epitopes could generate protection in guinea pigs and swine against the FMDV challenge. Recently, some recombinant TMV, such as TMVFN20 that contains an epitope FN20 from the FMDV VP1, were found to induce local necrotic lesions (LNL) on the inoculated leaves of a susceptible tobacco, Nicotiana tabacum Samsun nn. This hypersensitive-like response (HLR) blocked amplification of recombinant TMVFN20 in tobacco and limited the utility of recombinant TMV vaccines against FMDV. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigate the molecular mechanism of the HLR in the susceptible Samsun nn. Histochemical staining analyses show that these LNL are similar to those induced in a resistant tobacco Samsun NN inoculated with wild type (wt) TMV. The recombinant CP subunits are specifically related to the HLR. Interestingly, this HLR in Samsun nn (lacking the N/N'-gene) was able to be induced by the recombinant TMV at both 25°C and 33°C, whereas the hypersensitive response (HR) in the resistant tobacco plants induced by wt TMV through the N/N'-gene pathways only at a permissive temperature (below 30°C). Furthermore, we reported for the first time that some of defense response (DR)-related genes in tobacco were transcriptionally upregulated during HLR. CONCLUSIONS: Unlike HR, HLR is induced in the susceptible tobacco through N/N'-gene independent pathways. Induction of the HLR is associated with the expression of the recombinant CP subunits and upregulation of the DR-related genes.

An Ac transposon system based on maize chromosome 4S for isolating long-distance-transposed Ac tags in the maize genome.

Transposon tagging is an important tool for gene isolation and functional studies. In maize, several transposon-tagging systems have been developed, mostly using Activator/Dissociation (Ac/Ds) and Mutator systems. Here, we establish another Ac-based transposon system with the donor Ac tightly linked with sugary1 (su1) on maize chromosome 4S. Newly transposed Ac (tr-Acs) were detected based on a negative dosage effect, and long-distance-transposed Ac events were identified and isolated from the donor Ac by a simple backcross scheme. In this study, we identified 208 independent long-distance-transposed Ac lines. Thirty-one flanking sequences of these tr-Acs were isolated and localized in the maize genome. As found in previous studies, the tr-Acs preferentially inserted into genic sequences. The distribution of tr-Acs is not random. In our study, the tr-Acs preferentially transposed into chromosomes 1, 2, 9 and 10. We discuss the preferential distribution of tr-Acs from Ac systems. Our system is complementary to two other Ac-based regional-mutagenesis systems in maize, and the combined use of these systems will achieve an even and high-density distribution of Ac elements throughout the maize genome for functional-genomics studies.

The amplification and evolution of orthologous 22-kDa α-prolamin tandemly arrayed genes in coix, sorghum and maize genomes.

Tandemly arrayed genes (TAGs) account for about one-third of the duplicated genes in eukaryotic genomes. They provide raw genetic material for biological evolution, and play important roles in genome evolution. The 22-kDa prolamin genes in cereal genomes represent typical TAG organization, and provide the good material to investigate gene amplification of TAGs in closely related grass genomes. Here, we isolated and sequenced the Coix 22-kDa prolamin (coixin) gene cluster (283 kb), and carried out a comparative analysis with orthologous 22-kDa prolamin gene clusters from maize and sorghum. The 22-kDa prolamin gene clusters descended from orthologous ancestor genes, but underwent independent gene amplification paths after the separation of these species, therefore varied dramatically in sequence and organization. Our analysis indicated that the gene amplification model of 22-kDa prolamin gene clusters can be divided into three major stages. In the first stage, rare gene duplications occurred from the ancestor gene copy accidentally. In the second stage, rounds of gene amplification occurred by unequal crossing over to form tandem gene array(s). In the third stage, gene array was further diverged by other genomic activities, such as transposon insertions, segmental rearrangements, etc. Unlike their highly conserved sequences, the amplified 22-kDa prolamin genes diverged rapidly at their expression capacities and expression levels. Such processes had no apparent correlation to age or order of amplified genes within TAG cluster, suggesting a fast evolving nature of TAGs after gene amplification. These results provided insights into the amplification and evolution of TAG families in grasses.

Construction of a Coix BAC library and isolation of the 22 kDa α-coixin gene cluster.

Coix lacryma-jobi L. (Coix) is a close relative of maize and is considered a valuable genetic resource for crop improvement. Here we report the construction of the first Coix bacterial artificial chromosome (BAC) library using accession PI 324059. This BAC library contains about 230 400 clones with an average insert size of 113 kb, has low organellar DNA contamination, and provides 16.3-fold coverage of the genome. The library was stored in 12 × 96 pools that could be screened with a PCR protocol. Library screening was performed for the 22 kDa α-coixin gene family. A total of 57 positive pools were identified, and single clones were isolated from 19 of these pools. Based on DNA fingerprinting and Southern blot analysis, these 19 BAC clones form a single contig of about 340 kb in length, indicating that the 22 kDa α-coixin genes occur in a cluster. These results demonstrated the suitability of this BAC library for gene isolation and comparative genomics studies of the Coix genome.

Expression of the 26S proteasome subunit RPN10 is upregulated by salt stress in Dunaliella viridis.

Green algae of the genus Dunaliella can adapt to hypersaline environments and are considered model organisms for salinity tolerance. In an EST analysis in Dunaliella viridis under salt stress, we isolated a salt-inducible cDNA coding for the 26S proteasome subunit RPN10, designated DvRPN10. The DvRPN10 cDNA is 1472 bp and encodes a polypeptide of 377 amino acids. The DvRPN10 protein shares a high similarity to orthologs from other species. The function of DvRPN10 was confirmed by complementation of the yeast Deltarpn10 mutant. Q-PCR analysis of D. viridis cells grown in different salinities revealed that the transcript level of DvRPN10 increased in proportion to the external salinity within a range of 0.5-3 M NaCl, but decreased significantly at extremely high salinities (4-5 M NaCl). When a salinity shock of 1-3 M NaCl was applied to D. viridis cells, DvRPN10 mRNA levels remained steady during the first 36 h, and then gradually elevated to the level observed at 3 M NaCl. The gene structure of DvRPN10 was revealed by sequencing of a BAC clone containing this gene. Possible transcription factor binding sites related to stress tolerance were found in the promoter region of DvRPN10. The expression of DvRPN10 in response to the external salinity suggests that RPN10-mediated protein degradation plays a role in the salinity tolerance of D. viridis.

An expression analysis of 57 transcription factors derived from ESTs of developing seeds in Maize (Zea mays).

Maize seeds are an important source of food, animal feed, and industrial raw materials. To understand global gene expression and regulation during maize seed development, a normalized cDNA library, covering most of the developmental stages of maize seeds, was constructed. Sequencing analysis of 10,848 randomly selected clones identified 6,630 unique ESTs. Among them, 57 putative transcription factors (TFs) were identified. The TFs belong to seven different super-families, specifically 17 Zinc-finger, 13 bZIP, 8 bHLH, 6 MADS, 7 MYB, 3 Homedomain, and 3 AP2/EREBP. The spatial and temporal expression of the TFs was analyzed by semi-quantitative RT-PCR with representative tissue types and seeds at different developmental stages, revealing their diverse expression patterns and expression levels. One-third (19) of the maize TFs was found their putative orthologs in Arabidopsis. Similar expression patterns were observed in both maize and Arabidopsis for the majority of orthologous pairs (15 out of 19), suggesting their conserved functions during seed development. In conclusion, the systematic analysis of maize seed TFs has provided valuable insight into transcriptional regulation during maize seed development.

Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis.

Dunaliella, a unicellular green alga, has the unusual ability to survive dramatic osmotic stress by accumulating high concentrations of intracellular glycerol as a compatible solute. The chloroplastic glycerol-3-phosphate dehydrogenase (GPDH) has been considered to be the key enzyme that produces glycerol for osmoregulation in Dunaliella. In this study, we cloned the two most prominent GPDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively. Unlike higher plant GPDHs, both proteins contained extra phosphoserine phosphatase (SerB) domains at their N-termini in addition to C-terminal GPDH domains. Such bi-domain GPDHs represent a novel type of GPDH and are found exclusively in the chlorophyte lineage. Transient expression of EGFP fusion proteins in tobacco leaf cells demonstrated that both DvGPDH1 and DvGPDH2 are localized in the chloroplast. Overexpression of DvGPDH1 or DvGPDH2 could complement a yeast GPDH mutant (gpd1Delta), but not a yeast SerB mutant (ser2Delta). In vitro assays with purified DvGPDH1 and DvGPDH2 also showed apparent GPDH activity for both, but no SerB activity was detected. Surprisingly, unlike chloroplastic GPDHs from plants, DvGPDH1 and DvGPDH2 could utilize both NADH and NADPH as coenzymes and exhibited significantly higher GPDH activities when NADH was used as the coenzyme. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression. These results shed light on the regulation of glycerol synthesis during salt stress in Dunaliella.

Expressional profiling study revealed unique expressional patterns and dramatic expressional divergence of maize alpha-zein super gene family.

The alpha-zein super gene family encodes the most predominant storage protein in maize (Zea mays) endosperm. In maize inbred line B73, it consists of four gene families with 41 member genes. In this study, we combined quantitative real-time PCR and random clone sequencing to successfully profile the expression of alpha-zein super gene family during endosperm development. We found that only 18 of the 41 member genes were expressed, and their expression levels diverge greatly. At the gene family level, all families had characteristic "up-and-down" oscillating expressional patterns that diverged into two major groups. At the individual gene level, member genes showed dramatic divergence of expression patterns, indicating fast differentiation of their expression regulation. A comparison study among different inbred lines revealed significantly different expressed gene sets, indicating the existence of highly diverged haplotypes. Large gene families containing long gene clusters, e.g. z1A or z1C, mainly contributed the highly divergent haplotypes. In addition, allelic genes also showed significant divergence in their expressional levels. These results indicated a highly dynamic and fast evolving nature to the maize alpha-zein super gene family, which might be a common feature for other large gene families.