Gene networks and chromatin and transcriptional regulation of the phaseolin promoter in Arabidopsis.

The complete lack of seed storage protein expression in vegetative tissues and robust expression during embryogenesis makes seed development an ideal system to study tissue-specific expression of genes. The promoter for the Phaseolin (phas) gene, which encodes the major seed storage protein in bean (Phaseolus vulgaris), is activated in two sequential steps: Phaseolus vulgaris ABI3-like factor (Pv-ALF)-dependent potentiation and abscisic acid-mediated activation. In this study, a heterologous in vivo Pv-ALF/phas-GUS (for ß-glucuronidase) expression system in transgenic Arabidopsis thaliana leaves was used in conjunction with the powerful RNA-Seq approach to capture transcriptional landscapes of phas promoter expression. Remarkably, expression of over 1300 genes from 11 functional categories coincided with changes in the transcriptional status of the phas promoter. Gene network analysis of induced genes and artificial microRNA-mediated loss-of-function genetic assays identified transcriptional regulators RINGLET 2 (RLT2) and AINTEGUMENTA-LIKE 5 (AIL5) as being essential for phas transcription. Pv-ALF binding to the RLT2 and AIL5 promoter regions was confirmed by electrophoretic mobility shift assay. RLT2 and AIL5 knockdown lines displayed reduced expression of several endogenous seed genes, suggesting that these factors are involved in activation of endogenous Arabidopsis seed storage gene expression. Overall, the identification of these key factors involved in phas activation provides important insight into the two-step transcriptional regulation of seed-specific gene expression.

RNAi-mediated Ultra-low gossypol cottonseed trait: performance of transgenic lines under field conditions.

Cottonseed remains a low-value by-product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra-low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ-cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild-type levels of gossypol and related terpenoids. Although it was a relatively small-scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild-type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%-8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi-based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever-increasing needs of humanity.

Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination.

Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.

Sulfate and glutathione enhanced arsenic accumulation by arsenic hyperaccumulator Pteris vittata L.

This experiment examined the effects of sulfate (S) and reduced glutathione (GSH) on arsenic uptake by arsenic hyperaccumulator Pteris vittata after exposing to arsenate (0, 15 or 30 mg As L(-1)) with sulfate (6.4, 12.8 or 25.6 mg S L(-1)) or GSH (0, 0.4 or 0.8 mM) for 2-wk. Total arsenic, S and GSH concentrations in plant biomass and arsenic speciation in the growth media and plant biomass were determined. While both S (18-85%) and GSH (77-89%) significantly increased arsenic uptake in P. vittata, GSH also increased arsenic translocation by 61-85% at 0.4 mM (p < 0.05). Sulfate and GSH did not impact plant biomass or arsenic speciation in the media and biomass. The S-induced arsenic accumulation by P. vittata was partially attributed to increased plant GSH (21-31%), an important non-enzymatic antioxidant countering oxidative stress. This experiment demonstrated that S and GSH can effectively enhance arsenic uptake and translocation by P. vittata.

Transgenic expression of fern Pteris vittata glutaredoxin PvGrx5 in Arabidopsis thaliana increases plant tolerance to high temperature stress and reduces oxidative damage to proteins.

A glutaredoxin of the fern Pteris vittata PvGRX5 was previously implicated in arsenic tolerance. Because of possible involvements of glutaredoxins in metabolic adaptations to high temperature stress, transgenic Arabidopsis lines constitutively expressing PvGRX5 were evaluated for thermotolerance. Homozygous lines expressing PvGRX5 exhibited significantly greater tolerance to high temperature stress than the vector control and wild-type, based upon growth during stress and during recovery from stress, and this was related to leaf glutaredoxin specific activities. Measurements of tissue ion leakage, thiobarbituric acid reactive substances and protein carbonyl content showed that PvGRX5-expressors were significantly (P < 0.05) less affected by the high temperature treatment compared to wild-type and vector control lines for damage to membranes and proteins. Immunoblots indicated that specific protein bands, carbonylated during the stress treatment in the control lines, were protected in PvGRX5-expressors, thus implicating PvGRX5 in heat tolerance, likely mediated through cellular protection against oxidative stress.

Expression of a Pteris vittata glutaredoxin PvGRX5 in transgenic Arabidopsis thaliana increases plant arsenic tolerance and decreases arsenic accumulation in the leaves.

Chinese brake fern Pteris vittata hyperaccumulates arsenic in its fronds. In a study to identify brake fern cDNAs in arsenic resistance, we implicated a glutaredoxin, PvGRX5, because when expressed in Escherichia coli, it improved arsenic tolerance in recombinant bacteria. Here, we asked whether PvGRX5 transgenic expression would alter plant arsenic tolerance and metabolism. Two lines of Arabidopsis thaliana constitutively expressing PvGrx5 cDNA were compared with vector control and wild-type lines. PvGRX5-expressors were significantly more tolerant to arsenic compared with control lines based on germination, root growth and whole plant growth under imposed arsenic stress. PvGRX5-expressors contained significantly lower total arsenic compared with control lines following treatment with arsenate. Additionally, PvGRX5-expressors were significantly more efficient in their arsenate reduction in vivo. Together, our results indicate that PvGRX5 has a role in arsenic tolerance via improving arsenate reduction and regulating cellular arsenic levels. Paradoxically, our results suggest that PvGRX5 from the arsenic hyperaccumulator fern can be used in a novel biotechnological solution to decrease arsenic in crops.

Purification and characterization of thermostable monomeric chloroplastic Cu/Zn superoxide dismutase from Chenopodium murale.

Superoxide dismutase is the first line of defense against oxidative stress and thus helps in maintaining the cellular integrity. Chenopodium murale, a weed species adapted to widely varying climatic conditions faces extremes of temperatures ranging from 4 °C to 45 °C (Tmax) during growth and development. From this plant, we have purified a thermostable chloroplastic Cu/Zn superoxide dismutase (Chl Cu/Zn SOD) to homogeneity using minimal steps. Incubation of lysed chloroplasts at 70 °C for 1h reduced the interference of cytosolic SOD isoforms and reduced the protein content by 75 %. Chloroplastic SOD was purified from the heat stable fraction by gel filtration chromatography. The purified enzyme had a native molecular weight of 24 kDa, a half-life of 47.9 min at 80 °C and showed a single band at 24 kDa on SDS-PAGE. The N-terminus contained the conserved amino acids of chl Cu-Zn SOD. The Chl Cu/Zn SOD protein and its activity were enhanced under very high temperatures, high light intensities and in water stress/recovered C. murale plants under controlled environment conditions. Chl Cu/Zn SOD was also one of the predominant isoforms throughout growing period in field grown plants and declined during senescence. The Chl Cu/Zn SOD activity increased with the increase in ambient temperature and peaked in April with a 45 °C Tmax. These results clearly indicate that the chloroplastic Cu/Zn SOD is stably expressed at extreme environmental conditions. The presence of stable monomeric chloroplastic Cu/Zn SOD might help the plants to maintain the cellular homeostatis against adverse environmental conditions.

An arsenate-activated glutaredoxin from the arsenic hyperaccumulator fern Pteris vittata L. regulates intracellular arsenite.

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.

Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli.

Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI's role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction.