Aluminum-Induced Programmed Cell Death And Its Modulation By Signals In Yeast

Aluminum(Al) toxicity severely limits crop production on acidic soils, and therefore, Al toxicity and tolerance has been a research focus in terms of plant biology of adversity. Recent progress by a number of researchers has uncovered the molecular mechanism of Al exclusion by exudation of organic acid, but little is clearly understood about Al-resistance mechanism involving internal detoxification of Al. Gradually, some intriguing questions draw the attention of scientists such as: if programmed cell death(PCD) can be induced by Al in plants, if manipulation of the negative regulation process of PCD may provide a novel mechanism for conferring Al tolerance, how Al stress interact with signal transduction in vivo, and so on. For above questions, we made explorations by taking advantage of yeast- an excellent model for eukaryotic organisms- including its simple genetic background, shorter cell cycle, plenty of mutant strains, and conservatism during evolution. Results of my Ph.D. study can be summarized as follows:(1) Smiple procedures for yeast experiments were established through efforts, exemplified by transformation with plasmids using LiAc method, extraction of whole DNA by digestion with snailase, and devising instrument for facilitating time-course OD measurement by spectrophotometer. 1-4 mM Al was found to promote yeast growth, dramatically did 2 mM Al. The lower initial cell density in liquid medium, the more obvious the Al effect. Calculation by standard hemocytometer and flow cytometry indicates that Al actually accelerates cell division.(2) Observation of DAPI-stained nuclear shape under normal fluorescent microscope, TUNEL-stained DNA stand breaks under confocal laser-scanning microscope, external and internal characteristics of yeast cells under scanning electron microscopy and transmission electron microscopy, respectively, as well as measurement of DNA content by flow cytometry, together prove a process of PCD induced by Al.(3) The 3'-end and 5'-end cDNA fragments of Bax inhibitor-1 gene(BI-1) were successfully isolated from flowers of Phyllostachys praecox using RACE technique, and then sequence of a putative whole-length cDNA was obtained. In order to acquire a genuine open reading frame(ORF) of PpBI-1 for subsequent functional assay, PCR primers outside the putative ORF were designed, reverse transcripts of RNA were prepared as templates, and pfu polymerase was used to ensure sequence fidelity. (4) Collection of yeast strains and plasmids from oversea labs. Yeast strain EGY48 or BF264-15Dau was transformed with expression vector pGilda or pYX112 harboring PpBI-1, animal anti-apoptotic gene Ced-9 or Bcl-2. Afterwards, total DNA were extracted from yeast cells, and positive clones were selected by PCR method. Anti-LexA antibody was used to identify the anti-apoptotic members because of a LexA fusion part in pGilda. A fused fragment PpBI-1-GFP was constructed and then inserted into expression vectors for intracellular localization of LexA-PpBI-1-GFP, showing its distribution at perinuclear region. Data of growth, viability and PI permeability in either liquid or solid medium indicate that Ced-9,Bcl-2 or PpBI-1 can confer Al resistance to yeast cells. It's likely that Bcl-2 in yeast may exert its well-known dual function-inhibition of both PCD and cell cycle.(5) Anti-apoptotic members were found to remarkably prolong the duration for cold preservation, and to strengthen the growth-recovering ability of yeast cells. Based on this interesting phenomenon, preliminary research was conducted in relation to cold stress-induced apoptosis. As a result, chromatin circularization was found to emerge with high frequency under cold stress. Additionally, GSH deficiency resulted in augmented sensitivity of yeast cells to cold treatment, and the cold tolerance of PpBI-1 might be correlative to its synergistic action with GSH.(6) Flow cytometric studies using special fluorescent probes show that anti-apoptotic members can modulate Ca²⁺ flux but not ROS levels in response to Al stress. It is therefore likely that anti-apoptotic members probably function as modulators of calcium signaling, working downstream of ROS production or through ROS independent pathways. Depending on calcium sensitivity assay with mutant strains, a Ca²⁺/ATPase (Pmc1) -deficient strain K605 was found extremely sensitive to Al stress, which could be considered as entry to a novel research project. For the purpose of excluding non-specificity of K605 in response to stresses, a number of control experiments were performed. As a result, K605 was significantly sensitive to Al³⁺,H₂O₂ and Ca²⁺ , but neither to osmotic stress such as sorbitol and mannitol, nor to mental ions such as Cu²⁺ and Cd²⁺.(7) After plant expression vectors pCAMBIA-13011-PpBI-1 and pER8-PpBI-1 were constructed, transgenic Arabidopsis (T1) were screened out on hygromycin(Hyg)-contained B5 medium, and their seeds (T2) were well saved. Segregation ratios of seeds(T3) on Hyg-containing medium were statistically analyzed, and 1-3,2-1,2-2 and 3-9(T2) were thereby determined to be isogenic lines. Likewise, genome DNA was extracted to determine if PpBI-1 had been inserted into these lines, only line 1 and 2 being PpBI-1 positive. Therefore, seeds from 1-3 and 2-2(T3), 1-3-4 and 2-2-5(T4) were saved for further analysis. RT-PCR experiments show that seeds from 1-3-4(T4) can express a considerable amount of PpBI-1 even without induction by estrogen(Est), while seeds from 2-2-5(T4) must rely on Est in expressing PpBI-1, with relatively low efficiency.On basis of above work, we are facing novel questions and challenges, such as whether yeast Pmcl and its equivalents in plants are all potential Al-tolerant genes, how anti-apoptotic members modulate calcium signaling in yeast, so on and so forth. In summary, to investigate how to regulate PCD and calcium signaling would be helpful in understanding the mechanism of intracellular detoxification of Al in plants, and revealing a universal responsive mechanism under different stresses, which would finally make it possible to carry out the strategy of "One Gene Multiple Effects" in crop improvement against various kinds of pollution around.