Studies On Differences In The Tolerance To Cadmium Toxicity In Different Barley Genotypes And To Osmotic Stress Between Barley And Wheat Using Suspension Cell Cultures

Cadmium, one of the most toxic heavy metals in the environment, is easily absorbedand accumulated in plants. Excess accumulation of cadmium can seriously inhibit plantgrowth and development, and reduce the quantity and quality of crops. Furthermore, theaccumulation of cadmium do harm to human's health via food chains. Therefore, it is quiteimportant to understand the underlying mechanism of cadmium toxicity to plants and theresponse of plants to cadmium, for improving the quality of crops and inhibiting theinvolvement of cadmium in food chains. Up to now, most researches use the whole plant tostudy the biological effects of cadmium on plants. And it is unclear that how plant cellsrespond to the stress of cadmium. Suspension cells are highly homogeneous and moresensitive to stresses. Therefore, here the well-diffused and stable suspension cell lines weresuccessfully constructed for the two barley genotypes differing in cadmium tolerance. Thebiological effects of cadmium stress on antioxidant system of barley cells and the genotypedifference were systemically investigated by employing the newly constructed suspensioncell lines. The physiological and biochemical mechanism of alleviation and genotypedifference of Cd-toxicity by application of zinc, iron, GSH (reduced glutathione) and SA(salicylic acid) were performed respectively. In addition, the suspension cell lines of barley(PC1163) and wheat (PC988) were constructed. With these cell lines, the influence of salt(NaCl treated) and desiccation (PEG treated) stresses on cell growth and cellular prolinecontent were investigated, and the underlying response mechanisms were explained basedon transcriptional changes with or without the stresses. The main results were summarizedas follows:1. Stable and homogeneous suspension cell lines of two barley genotypes (Weisuobuzhiand Dong 17) were successfully constructed, and the growth characteristics of cell lineswere investigated. In addition, the effects of different cadmium levels on cell viability werealso tested. Three phases in the growth of the barley suspension cell lines were found,including the lag phase (0-3 d), logarithmic phase (4-8d) and stationary phase (9-12d), indicating the best subculture cycle of barley suspension cells is 7-8 days. The pH valuechanges of the cell lines depended on the growth phase: an acute decrease firstly, a slowincrease secondly, and a modest decrease finally. Cell viability decreased with the increaseof treating time and cadmium level. A treatment condition, 50μM cadmium for 5 days, wasfound suitable for performing a further investigation of physiological and biochemicalresponse of cell lines, by comparing the cell viability and MDA content.2. Cell viability and the activities of antioxidant enzymes in the two cell lines ofWeisuobuzhi and Dong17 under cadmium stress with addition of a serial concentration ofzinc or iron were investigated. It was found that cadmium stress induced a significant lossof cell viability and MDA accumulation. And the sensitive genotype Dong 17 was foundmore sensitive to the stress than the cadmium-tolerant cell line Weisuobuzhi. With the stressof 50μM cadmium, SOD and POD activities in tolerant line Weisuobuzhi were remarkablyinduced, but CAT activity was repressed significantly. SOD and CAT activities of thesensitive cells Dong 17 increased strongly after treated with cadmium for 1 day, butdecreased in 5 days after treatment. However, POD activity in the treated cells was higher,compared to the untreated control. With the cadmium treatment, the absence of zinc or ironnot only aggravated the loss of barley cell viability and the increase of cellular MDA, butalso reduced the activities of SOD, POD and CAT. Interestingly, either 300μM zinc or 500μM iron could relieve the oxidative damage of cadmium and improve the activities ofantioxidant enzymes and reduce the cellular MDA accumulation.3. We investigated the effect of exogenous GSH or SA on antioxidant system of the twocell lines of Weisuobuzhi and Dong17 under cadmium stress. It was found that 50μMcadmium can induce the activities of APX and MDHAR in the tolerant strain Weisuobuzhiand also make the activities of GR and DHAR significantly increase in later phase aftertreatment. However, the same treatment only enhanced MDHAR activity in the sensitivestrain Dong 17. In the later phase after treatment, GR activity increased a bit but APXactivity significantly decreased, while DHAR activity was repressed in the whole period.Although 50μM cadmium reduced GSH and AsA contents in the both cell lines, the cellular GSH and AsA contents and GSH/GSSG and AsA/DHA ratios in cadmium tolerant cellswere found higher than that in the sensitive one. Both of GSH and SA could reduce thecadmium-induced oxidative damage and make a lower content of cellular MDA comparedto the single treatment of cadmium, and also increase the cell viability. The exocellularGSH improved APX and MDHAR activities and enhanced the cellular GSH content andGSH/GSSG ratio in both cell lines. Similarly, SA also increased the cellular GSH contentand GSH/GSSG ratio in both cell lines, and improved the activities of the antioxidantenzymes, enzymes involved in the AsA-GHS cycle and the cellular ASA content andAsA/HDA ratio in the cadmium-tolerant cell lines.4. Here, the effect of salt (NaCl treated) and drought (PEG treated) stresses on cellgrowth and cellular proline content were investigated using the suspension cells of barley(PC1163) and wheat (PC988), and the underlying mechanisms of the difference in osmotictolerance between barley and wheat were also explained basing on transcriptional changes.It was found that the osmotic tolerance of barley cells was stronger than that of wheat cells.After 24 h treatment of 100mM NaCl, the cell viability of wheat (PC998) significantlydecreased but barley cells did not. Proline is one of most important osmotic regulators inGramineae. The proline content in wheat cells is nearly 3 times higher than that in barleycells. The cellular proline contents in both cell lines were enhanced with increasing timeunder NaCl stress, however, it did not changed significantly with the treatment of PEG.With the treatments of NaCl and PEG, the transcriptional changes of two key genes (P5CSand P5CR) involved in proline synthesis were measured by RT-PCR and found to becorrelated to the cellular proline content in wheat cell lines. However, the transcriptionallevels of both genes did not significantly change in barley cells, but the proline content wasenhanced with increasing time. Furthermore, we found that the transcriptional levels ofboth genes in barley cells were higher than these in wheat cells, but the cellular prolinecontent in barley cells was found obviously lower than that in wheat cells. These resultssuggested that the synthesis of cellular proline in barley is higher than that in wheat cellsand the cellular proline in barley maintains a high level of flux. The result was also confirmed by measuring the transcriptional levels of four hordein homologous genes in thebarley cells. Free proline participates in the synthesis of B3-Hordein, which plays a key rolein regulating the balance of the cellular proline.