植物生理与分子生物学学报最新文献

Sulfite oxidase (SO), one of the known molybdenum co-factor-containing enzymes, plays important roles in diverse metabolic processes such as sulfur detoxification and purine catabolism in mammals. But much less is known about the expression and regulatory characterization of sulfite oxidase gene in higher plants. In this report, expression of Arabidopsis SO is characterized in detail by semi-quantitative RT-PCR and histochemical staining. The results showed that the transcripts of AtSO were predominantly detected in Arabidopsis aerial tissues including stems, young leaves, young inflorescences and immature siliques at higher level, but in roots with a lower level. To monitor AtSO expression in plant, the promoter region containing a 1 562-bp genomic sequence from AtSO was isolated and analyzed using methods of bioinformatics. Basing on the distribution of beta-glucuronidase (GUS) activities shown by histochemical staining in transgenic Arabidopsis plants harboring the promoter-uidA fusion construct, it can be concluded that AtSO is expressed mainly in the green tissues/organs in a light-dependent way. In addition, its expression is up-regulated during sulfite treatment. The information from this study may provide useful clue for further functional analysis of plant SO homologs during light-induced development of leaf tissue and/or excessive sulfite/SO(2) gas stresses in higher plants.

Differential expression of the key genes controlling phenolic metabolism in allelopathic and non-allelopathic rice accessions was investigated under two nitrogen supply levels (lower and normal) using fluorescence quantitative-polymerase chain reaction (FQ-PCR) (Figs.2, 3). The results indicated that 9 key enzyme genes concerned were mediated by lower nitrogen level (Table 2). All of the nine genes (Table 1, Fig.4), were up-regulated by 1.9-5.4 times of the relative gene expression amounts in allelopathic rice accession, 'PI312777' under the lower nitrogen condition compared with their controls, of which PAL gene showed the highest relative gene expression amount with 5.4 times of the relative gene expressions compared with the control, while in non-allelopathic rice Lemont, seven genes were down-regulated by 29%-72% under lower nitrogen supplies compared with their controls and only two genes, i.e., phenylalanine ammonia-lyase and cinnamoyl-CoA genes were up-regulated, which however were a decrease of 22% and 74% over those in allelopathic rice accession (Table 2). These findings strongly suggest that the increase of allelopathic potential induced by 1/4 nutrient stress was responsible for enhanced phenolic compound synthesis metabolism.

Previous data showed that a 31-bp (from -840 bp to -810 bp) DNA fragment located at the 5' upstream region of rice waxy gene could interact with nuclear protein extracted from developing endosperm of rice. When this 31 bp DNA sequence was used as a bait to screen a rice cDNA library with a yeast one-hybrid system, three groups of cDNA clones were isolated. One of them is pC73, the correspondent rice gene of pC73 was named as OsBP-73 (Oryza sativa binding protein). A pull-down assay was made to identify the target genes of transcription factor by using genomic DNA and recombinant p73 protein. The cDNA fragment containing DNA-binding domain of OsBP-73 was cloned into expression vector pET28-c(+) (Fig.1) to produce protein p73, fused with a his(6)-tag, from E. coli BL21 (DE3) (Fig.2). The p73 was purified with Ni-NTA under native condition (Fig.3). The target genes of p73 were identified in rice genome-wide by using a pull-down assay, and 22 candidate genes were obtained (Figs.4 and 5, and Table 1). The obtained results show that putative light-repressible receptor protein kinase and GAMYB-binding protein could serve as targets of the OsBP-73, suggesting that OsBP-73 might be involved in light signal transduction.

Gamma-tubulin gene is a very important house-keeping gene and plays key roles in cell division, microtubule nucleation and cell cycle regulation in both plants and animals. In this paper, we studied the gamma-tubulin gene silencing in Nicotiana tabacum var. Samsun NN using the recombinant PVX vector with D fragment of gamma-tubulin cDNA. Special phenotype appeared from recombinant PVX-vector-infected plants, the different morphological leaves grew alternatively in leaf layers (Figs.3-5), and all the buds fell off gradually (Fig.4), resulting in failure of all the microspores to develop to the tetrad stage (Fig.8). The silencing initiated from the apical tissue and kept on until it was mature (Figs.3, 4). There was the increasing of target gene mRNA obviously during gene silencing, even to exceed the positive control (Fig.6). The PVX vector was also suppressed strongly and could fluctuate to some degree (Fig.9), which was probably correlated with target gene mRNA expression.

The total photosynthetic electron flux through PSII [J(e) (PSII)], the electron flux used for carbon assimilation [J(e) (PCR)], the electron flux used for photorespiration [J(e) (PCO)], the electron flux used for Mehler reaction [J(a) (O(2)-depend)] and the electron flux used for nitrogen metabolism [J(a) (O(2)-independ)] in leaves of Rumex K-1, a fodder crop with high protein content, were measured under three levels of nitrogen application (Fig.2). The nitrate reductase (NR) activity, glutamine synthetase (GS) activity, the leaf protein content, the chlorophyll content, P(n) and Phi (PSII) and F(v)/F(m) (Table 1) were also measured. The results showed that with the increase of nitrogen application, the NR and GS activities increased remarkably (Fig.3) and more electron flux was allocated to nitrogen metabolism as well as photorespiration (Fig.2). Nitrogen metabolism and carbon metabolism competed for energy, and the proportion of energy used in nitrogen metabolism to that used in carbon metabolism changed with nitrogen application rate. The electron flux used for nitrogen metabolism is about 15%-21% of the total electron flux under the three levels of nitrogen application (NO(3)(-) 0-30 mmol/L). Under lower nitrogen application, though energy used for carbon and nitrogen assimilation remarkably decreased, no significant increase of electron flux allocated to Mehler reaction was observed. The excess excitation energy in the leaves under the lower nitrogen application was efficiently dissipated via other energy dissipation mechanisms to protect the leaves against photo-damage.

H(2)O(2), plasma membrane H(+)-ATPase (PM H(+)-ATPase) and salicylic acid (SA) play important roles in sensing external stimulation and activating defense responses in plants. However, it remains uncertain whether they are involved and interrelated in response to heat acclimation. Experiments were performed by pharmacological methods, and the relationship and the connection between endogenous H(2)O(2), free SA and PM H(+)-ATPase were investigated in pea plants (Pisum sativum L.) during heat acclimation. The results showed that an accumulation peaks of H(2)O(2), free SA and PM H(+)-ATPase, were detected during heat acclimation at 37 degrees C for 2 h and H(2)O(2) burst appeared before SA accumulation that followed by increase of PM H(+)-ATPase activity (Fig.1). Pretreatments with either scavengers of active oxygen species (dimethyl sulfoxide and ascorbic acid) or antioxidant (reduced glutathione) inhibited the increases in both H(2)O(2) and free SA contents as a part of heat acclimation (Fig.2). Additionally, changes in activity of plasma membrane NADPH oxidase paralleled with H(2)O(2) level during heat acclimation (Figs.1 and 3), implicating that H(2)O(2) might be generated by plasma membrane NADPH oxidase. Moreover, pretreatments with either diphenylene iodonium (DPI), a suicide substrate inhibitor of plasma membrane NADPH oxidase, or dimethylthiourea (DMTU), a quencher of H(2)O(2), could block the increase in free SA content and activity of plasma membrane NADPH oxidase as a part of heat acclimation (Fig.4). According to the assay described above, it is suggested that both H(2)O(2) and PM H(+)-ATPase participate in SA signaling that leads to the development of thermotolerance in pea plant, and H(2)O(2) functions upstream and PM H(+)-ATPase functions downstream of the SA signal. Also, the regulation mechanism of PM H(+)-ATPase activity was investigated, which showed that during heat acclimation, increase of PM H(+)-ATPase activity was independent of PM H(+)-ATPase amount and the enzyme activity may be modulated at post-translational level that may involve in reversible protein phosphorylation (Fig.5).

The effects of G protein regulators and stylar S-RNase on the growth and [Ca(2+)](i) changes of the Pyrus pyrifolia pollen tube were investigated using Laser Confocal Scanning Microscope (LCSM). The results indicated that: (1) The growth of 'Housui' pollen tube could be inhibited by its stylar S-RNase and pertussis toxin (PTX), the inhibitor of heterotrimeric G protein. While 'Kousui' stylar S-RNase had little effect on the growth of 'Housui' pollen tube; cholera toxin (CTX), the activator of heterotrimeric G protein, could promote pollen tube growth and eliminate the suppression of stylar S-RNase on the growth of self-pollen tube, but the growth of 'Housui' pollen tube could be arrested under the co-action of PTX and S-RNase from 'Kousui' Fig.1). (2) Treatments with different stylar S-RNase and G protein regulators could have different effects on the change in [Ca(2+)](i) in the tip of pollen tube (Figs.2,3). The treatment with 'Housui' stylar S-RNase could induce the decrease of fluorescence gradient of [Ca(2+)](i) along the tip of self-pollen tube (Figs.2A, 3A), and the treatment with CTX could markedly elevate [Ca(2+)](i) in the tip of pollen tube showed (Fig.3C). The way of [Ca(2+)](i) changed in 'Housui' pollen tube under the co-action of CTX and its stylar S-RNase showed the compositive effect of the two respective treatment (Fig.3A, C, E), but the effect of the co-action of PTX and 'Kousui' stylar S-RNase showed increase in [Ca(2+)](i) in the pollen tube in 18 min after treatment, and then decrease between 18-36 min (Fig.3F). These results suggest that during self or cross pollination, the control of the growth of Pyrus pyrifolia pollen tube is by the synergistic effect of stylar S-RNase, G protein and [Ca(2+)](i) in the pollen tube.

A simple protein in-gel digest method compatible with MALDI-TOF MS analysis was developed from the previously reported protocols with the following modifications. (1) The washing step was intensified by increasing the volume of distilled water and prolonging the vortex time. (2) Trypsin was acidified to improve the enzyme activity. (3) A Ca(2+) free pre-digest solution was used to reduce the trypsin auto cleavage. (4) The procedure of removal of salts and SDS was cancelled. (5) The digest was directly used to perform MALDI-TOF MS analysis. The compared results of this method with a latest reported protocol demonstrated that the method could efficiently reduce the peptide loss, provide more information for MS analysis, and consequently make the protein identification more reliable.

As the key regulators of cell wall extension during plant growth, expansins play an important role in regulating the development and response of plants to adverse environment. The characteristics of expansins in wheat coleoptiles and their responses to water stress were studied. Expansin proteins were extracted from wheat coleoptiles by the methods of Hepes or SDS. The activities of expansins were measured with an improved extensometer and the amount of expansins was measured by immunoblot analysis with the expansin antibody. The results showed that in coleoptiles, the extension of native cell walls depended on acidic pH, and the expansins were found to be located at cell walls by location analysis. Expansins from wheat coleoptiles could induce cell wall extension both of cucumber hypocotyls and coleoptiles, and vice versa, albeit with differences noted in extension activity. The changes in activity and abundance of expansins in wheat coleoptiles in response to water stress suggest that expansins may play a significant role in the tolerance of wheat plants to water stress.

The expressions of BtCry1Ac insect-resistance genes and rhizogenesis genes and their response to NaCl stress were studied using tissue culture plants of high transgenic insect-resistant 'poplar 741' and transpolygenes 741 (insect-resistant genes and T-DNA of Ri plasmid). The results showed that IAA and GA contents increased quickly, plant root number increased and root length reduced after rhizogenesis and hormone synthesis related gene in Ri T-DNA were inserted into the genome of poplar (Figs.2, 3, 8 and 9). Plant height, root number, chlorophyll content, IAA and GA contents decreased gradually with an increase in NaCl stress intensity (Figs.1, 2, 4-6, 8 and 9). Apiece index change extent of transgenic rol gene plant was smaller than transgenic Bt gene plant and non-transgenic plant. Bt toxin protein content of transgenic rol gene plant increased significantly under NaCl stress (Fig.7). Our results indicate that the expressions of the foreign genes changed with the changes of the environmental conditions.