New Negatives in Plant Science最新文献

Background

Aspartate aminotransferase (AAT) catalyzes a reversible transamination reaction, producing aspartate and 2-oxoglutarate from glutamate and oxaloacetate, in various cellular plant compartments. Previous work in our lab had shown that a similar aminotransferase enzyme, alanine aminotransferase (AlaAT), produced nitrogen use efficient (NUE) phenotypes when over-expressed in canola (Brassica napus) under the salt-stress inducible promoter, btg-26. Given the similarities between these two enzymes and their roles in plant metabolism, it was hypothesized that over-expression of AAT could also produce an NUE phenotype in canola.

Results

Transgenic Brassica napus lines over-expressing AAT from Medicago sativa were produced and analyzed for NUE phenotypes under both high and low nitrogen conditions. While several lines showed promising increases in biomass under the various fertilizer regimes, these alterations could not be reliably replicated and increases in expression of the transgene detected via RT-PCR did not translate into significant increases in AAT activity in plant tissues.

Conclusions

Transgenic Brassica napus lines over-expressing AAT do not display NUE phenotypes similar to those plants over-expressing AlaAT. Although this work produced a negative result, it is important to compare the NUE phenotype produced by over-expression of AlaAT and AAT, and differences in metabolism between AAT vs AlaAT over-expressing lines which may be used to deduce changes in plant N metabolism important for NUE in cereal crops.

Background

Expansin has been proposed to be an enhancer of cellulase activity in the deconstruction of biomass for sugars for industrial applications. However, the expansin protein is present in plant tissue only in minute quantities for promoting growth. Thus, producing adequate amounts of expansin for applications in industry will require a heterologous system that will over-express an expansin gene to produce large quantities of expansin protein. Development of a production system requires a facile, rapid assay. However, because no straightforward assay for expansin protein exists, we attempted to make milligram quantities of the protein in a fast or transient system for anti-expansin antibody preparation for use on Western blots or in ELISA assays.

Results

We tested the expression of the cucumber expansin gene in several heterologous systems including Escherichia coli and transient Nicotiana benthamiana leaves with limited success. We also had limited success in transiently expressing an alternative expansin gene from bamboo in N. benthamiana leaves. In order to determine if expansin over-expression is limited to a seed system, Arabidopsis thaliana seeds were tested. Although all positive and negative controls behaved as expected, none of these common systems expressed the expansin gene well.

Conclusions

Over-expression of cucumber expansin in three heterologous systems, E. coli, transient tobacco leaves, and Arabidopsis seeds was unsuccessful. The cause of this failure is not known. These results confirm the necessity of experimentally exploring several heterologous systems for protein production in order to find one with utility.

Background

CaM-like proteins (CMLs) are localized in the cytosol and others in organelles such as the mitochondria, the peroxisomes and the vacuole. To date, although several plastidial proteins were identified as CaM/CML interactors, no CMLs were assigned to the chloroplast. Absence of clues about the genetic identity of plastidial CMLs prevents investigating their regulatory role.

Results

To improve our understanding of plastidial Ca²⁺ regulation, we attempted to identify plastidial CMLs with two large scale, CaM-specific proteomic approaches, and GFP-fusions.

Conclusions

Despite the use of several different approaches no plastidial CML could be identified. GFP fusion of CML 35 CML36 and CML41 indicate a cytosolic localization.

Background

Switchgrass (Panicum virgatum) is one of the leading candidates to provide lignocellulosic biomass for biofuel production. Switchgrass is capable of relatively high productivity on marginal land or when intercropped with trees. Production of switchgrass is dependent upon light use efficiency at the canopy level. Thus, maintenance of photosynthesis at light limiting and cool conditions ought to elongate the growing season and increase productivity of switchgrass. Photosynthesis under cool conditions and low light is maintained higher in giant miscanthus (Miscanthus × giganteus) than switchgrass by retaining relatively high expression of pyruvate orthophosphate dikinase (PPDK). Our main goal was to create lines of switchgrass with upregulated PPDK and to evaluate photosynthetic responses of those lines to growth temperature under low radiation conditions. Our approach was to grow replicate plants of each transgenic event with an untransformed control in low light environments at either warm (28 °C day/24 °C night) or cool (14 °C day/12 °C night) conditions. Photosynthesis parameters of all plants were assessed with fluorescence kinetics, light response curves and carbon dioxide response curves.

Results

We created several lines of transgenic switchgrass with documented upregulation of cDNA for the PPDK gene (C4ppdk1). Photoinhibition was higher in the transgenic lines, but electron transport rates (ETR) and quantum yield of photosystem II were not inhibited by cool conditions. The higher than expected ETR under cool conditions was associated with increased non-photochemical quenching, which indicated that enzymatic reactions of photosynthesis were inhibited more by cool conditions than photochemical processes. In all except one transgenic line, most metrics of biochemical processes decreased under cool growth conditions, which resulted in significantly lower productivity under cool conditions.

Conclusions

All transgenic lines were able to balance electron transport and biochemical process at low radiation keeping apparent quantum yield constant and the light saturation point relatively low. Thus, the photosynthetic changes associated with the transgenic events could make the transgenic lines appropriate for use in low light regions such as forest intercropping systems if productivity was increased. Although one transgenic line had weakly improved photosynthesis under cool conditions in this study, improving cold temperature photosynthesis in switchgrass will require more than manipulating the expression of a single gene.

Background

Abscission of young fruitlets is a widespread phenomenon in fruit trees termed ‘physiological fruitlet drop’ (PFD). For some fruit crops, the rate of PFD is agriculturally sufficient, and in many cases too intense. In apples (Malus domestica) PFD is insufficient since without additional fruitlet thinning, fruits will not reach commercial size and trees will enter a cycle of alternate bearing. An apple inflorescence contains 5–6 flowers, the terminal king flower is the first to initiate, the first to reach anthesis, and is considered the fruitlet with the lowest chance to go through PFD. The last flower to initiate and later reach anthesis is termed lateral 1 (L1), and it has the highest probability to enter PFD. A better understanding of the PFD process might lead to more precise thinning procedures. The current hypothesis is that the ‘sink strength’ of the L1 fruitlet is weak compared to king fruitlet, thus L1 will enter a nutritional shortage which will lead to its developmental arrest and abscission. Based on this hypothesis, we assumed that the concentration of carbohydrates (soluble sugars and starch) would be highest in king and lowest in L1.

Results

We traced the level of different soluble sugars and starch in the different flowers/fruitlets in the apple inflorescence, before anthesis and during the early development of the apple fruitlet. Sugar levels were indeed higher in king compared to L1 at initial stages, even before anthesis. While this result nicely fit the consensus hypothesis, we considered it might be an artifact caused by the different developmental stages reached by each flower within the inflorescence. When we normalized the collection of different flowers within an inflorescence to a certain developmental stage and not to a date, the differences in sugar level were reduced or non-existent.

Conclusions

While carbohydrates are clearly essential for young fruitlet survival, our finding suggests that the ability of L1 fruitlets to collect sugars is not reduced compared to other fruitlets in the cluster, just slightly delayed. If a nutritional shortage indeed occurs in L1 fruitlets, the type of chemical in shortage, or the cells that suffer from this shortage, are still unknown.

Background

Scientific efforts directed toward improving the defense of plants to pathogens are dependent on knowing how the defense responses are signaled. In general a given plant can resist essentially all challenging pathogens except for the true pathogens which have developed means to suppress or evade the plant's “non-host” resistance response. Thus understanding the signaling of this potent level of immunity is paramount.

Findings

The initiation of transcription of defense genes associated with non-host resistance responses in plants has been hypothesized both as the direct targeting of DNA (chromatin) and as an indirect activation of transcription factors following a PAMP (effector)/PRR (receptor) recognition. Documentation exists for both routes and this report evaluates two PAMPs in the pea endocarp system in which DNA damage has been proposed to directly initiate defense gene transcription. The induction of immune responses resulting from direct effects on chromatin has not received the attention warranted. To account for flexibility in initiating transcription of defense genes, the plant must be responsive to challenges by every organism and all biological elicitors. The PAMP/PRR hypothesis is visualized as families of pattern recognition receptor proteins localized mainly in the cell membrane that become bound with effectors thereby cascading a signal to specified transcription factors. Direct challenges from nuclear penetrating elicitors in the pea non-host resistance system are visualized as causing an unlimited diversity of structural changes to chromosomal regions in the vicinity of plant non-host defense genes and subsequently enhancing their transcription. This report further evaluates the action of candidate PAMPs; flg22 from bacteria and chitin from fungi, in signaling the non-host resistance response of peas.

Conclusions

The results indicate that these PAMPs only marginally elicit pisatin production compared with challenges containing intact fungal spore suspensions. High external conc. of the PAMPs activated both a set of PR (pathogeneses-related) genes and developed cytological-detectable disease resistance against a true pea pathogen. The failure of lower concentrations of chitinous treatments to activate cytological detectable disease resistance, PR gene induction or pisatin accumulations suggests the existence of additional signaling routes to the non-host disease resistance generated by intact fungal spores. DNA damage to the pea DNA was detected indicating direct effects from PAMPs on the chromatin occur. Only high concentration levels of these PAMPs appear to have the potential to constitute a portion of those signals generating responses in peas against the vast arsenals of microbes in nature.

Background

It has been said that naturally occurring autopolyploid strains are more tolerant of biotic and/or abiotic stresses, due at least in part to the higher accumulation of secondary metabolites. Data supporting this hypothesis come from comparisons between naturally established autopolyploids and diploids; thus the high accumulation of metabolites in polyploid strains may be a secondarily acquired feature and not a direct effect of the autopolyploidy. But no detailed studies on this issue have been carried out.

Results

Here we carried out metabolome analyses between newly created tetraploids and the parent diploid in a model plant, Arabidopsis thaliana, and the agriculturally important pear fruit tree (Pyrus communis var. sativa). Our data showed that small numbers of metabolite species differ in amount between diploids and tetraploids in both species, but the differences were not reproducible among growth conditions and species.

Conclusions

These results strongly indicate that metabolite content is not universal nor the direct target of polyploidy-dependent changes. Instead, naturally occurring hyperaccumulation of metabolites in autopolyploids may be the result of secondary natural selection.

Salinity is an important abiotic factor that limits crop productivity for which solutions are being investigated through extensive research in plant biotechnology. The current study was designed to determine if expression of Escherichia coli nhaA gene, a Na⁺/H⁺ antiporter, can confer salt tolerance in transgenic rice cultures and plants. Transgenic rice calli and plants containing the nhaA gene were treated with various concentrations of NaCl. Lower biomass and higher death rates were observed under salt stress conditions for these transgenic materials. These data suggest that transgenic rice containing the nhaA gene driven by the maize ubiquitin promoter are salt sensitive. In contrast to previous reports, an enhanced salt tolerance after nhaA expression in rice cultures and plants is not demonstrated in our current study.

Background

The chloroplast genome of plants has been frequently sequenced using chloroplast DNA derived by techniques involving chloroplast isolation and or by PCR amplification using primer sequences targeted to amplify the chloroplast genome. Using these approaches, chloroplast heteroplasmy, described as variations in the chloroplast sequence within an individual plant, has been reported in many plant species. More recently, next generation sequencing (NGS) technologies have allowed chloroplast genome sequences to be extracted from shotgun sequences of total plant DNA.

Results

Here, we used DNA preparations varying in nuclear, mitochondrial and chloroplast enrichment to explore the potential to distinguish genuine chloroplast heteroplasmy from apparent heteroplasmy due to sequence-variant homologues of chloroplast genome sequences inserted in nuclear or mitochondrial genomes. Application of NGS to the whole sugarcane genome followed by read mapping analysis of the complex sugarcane system allowed the assembly of a complete chloroplast genome sequence of sugarcane cv. Q155. Variant analysis showed that they were present only at frequencies that could be attributed to homologues of chloroplast sequences inserted in the nucleus or mitochondria.

Conclusions

The result suggests that earlier reports of heteroplasmy in chloroplasts may have been due to contaminating sequences from other genomes (nuclear or mitochondrial) in chloroplast preparations or specific amplification of sequences from these genomes. This demonstrates that the ability to evaluate sequence abundance avoids the risks of attributing a chloroplast gene homologue from the nucleus or mitochondria to the chloroplast.

Background

Stevia rebaudiana Bertoni is an important anti-diabetic medicinal herb containing non-caloric sweet compounds. In this study, the effect of gamma irradiation on growth kinetics and accumulation of various bioactive compounds were investigated during callogenesis.

Results

Callus was developed from leaf pieces inoculated on Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l⁻¹), α-naphthalene acetic acid (NAA), indole butyric acid (IBA) and gibberellic acid (GA₃; 0.3 mg l⁻¹). After 30-days, vigorous calli were transferred to fresh medium and exposed to various gamma irradiations (5.0, 10, 15 and 20 Gy). It has been observed that the increasing doses of gamma rays inhibited callus proliferation (88.61–79.16%) as compared to control (95.83%). Similarly, 10, 15 and 20 Gy doses induced friable, granular and spongy callus as compared to control (compact). Furthermore, 5.0, 10 and 20 Gy doses significantly reduced the fresh callus biomass (FCB), however, 15 Gy dose enhanced FCB (1660 mg) and dry callus biomass (DCB; 159.36 mg) than control (1520; 145.92 mg). The chromatographic data revealed that 15 Gy dose slightly enhanced stevioside content (0.251 mg/g-DCB) than control (0.232 mg/g-DW), while other doses showed a negative effect on stevioside content. Higher antioxidant activity (88.73%) was observed in 20 Gy treated callus cultures. However, higher total phenolic content (TPC; 43.90 mg/g DCB) and total flavonoids content (TFC; 6.87 mg/g DCB) were observed in 15 Gy treated callus cultures.

Conclusions

The application of gamma irradiation did not show major variation in biomass and bioactive compounds production in callus cultures of S. rebaudiana.