Genotypic Difference In Arsenic Uptake, Subcellular Distribution And Speciation In Tobacco And The Possible Role Of Phosphate In Alleviating Arsenic Toxtcity

Arsenic (As) is a metalloid that is highly toxic even in small amounts. Various environmental processes which include utilization of As compounds such as fertilizers, pesticides and desiccants have hastened the rapid accumulation of As in the soil. China is the most leading world tobacco producer. In order to alleviate the effects of As, it is of due importance to select tobacco genotypes that are tolerant to As, so that they produce the required yields of tobacco even under high As concentrations. In addition, understanding the distribution of metals in subcellular parts is also of necessity in order to know why some plants are more affected by As than others. Arsenic accumulation and toxicity in plants differs greatly among plant species and also among organs or tissues in the same plant. Therefore to understand the difference in As toxicity among plant tissues, it is necessary to study its form and subcellular distribution. Although As is highly toxic and provides no essential benefit to plants, the chemical similarity between arsenate (As5+) and phosphate (PO3-4) enables PO3-4to suppress As5+uptake and hence inhibits the disastrous effects of As on plants. The aims of this study were to (1) investigate the difference in As accumulation among three different tobacco genotypes;(2) to understand the genotypic differences in As accumulation relevant to its distribution in various subcellular plant parts in two different As accumulating tobacco genotypes; and (3) to investigate the differences in As5+and PO43-uptake mechanisms and to evaluate the effects of external PO43-in controlling As5+uptake in tobacco genotypes differing in As sensitivity. The results are as follows:1. Arsenic uptake and accumulation dynamics in different tobacco(Nicotiana tabaccum) genotypes under varying arsenic treatmentsThe effects of As were studied in a hydroponic experiment using3tobacco genotypes, ZY90, Y85and FSMY differing in As tolerance. The plants were treated with0,10,20,40and80μM As and samples were collected after48and96hours after treatment. Shoot and root dry weights were reduced in a dose and duration dependent manner, with the greatest decrease being observed at80μM after96hours of treatment in ZY90. The decrease in shoot and root dry weight followed the trend ZY90>Y85>FSMY. ZY90had higher internal As concentration than Y85and FSMY only at10,20and40μM As concentration while FSMY had higher internal As concentration at80μM As. However, FSMY had the greatest As accumulation and the least As sensitivity at all time intervals, while in contrast ZY90had the least As accumulation and the greatest As sensitivity. The results revealed that although FSMY is a high As accumulating genotype, it is less sensitive to As, suggesting that it does not achieve its tolerance by restricting As uptake, but by another mechanism.2. Arsenic subcellular distribution and speciation in tobacco genotypes with different arsenic toleranceSubcellular distribution of As was investigated in a hydroponic culture using2different As tolerant tobacco(Nicotiana tabacum) genotypes ZY90and FSMY. Four As treatments viz0,1,10and100uM were applied and As concentration was measured in5different fractions, cell wall, plastid, nucleus, mitochondria and soluble fraction. The results showed that in root, As was mainly localized in cell walls whereas in stem and leaf As was localized in both cell wall and vacuoles. This is evidence that both the cell wall and vacuoles are major barriers in protecting plants against As toxicity. A distinct difference between genotypes in As concentration in subcellular and chemical forms was found with the As-sensitive genotype ZY90containing higher As in plastid, nucleus and mitochondria and lower As in cell wall and soluble fraction in comparison to FSMY. Arsenic speciation analysis was carried out employing liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) and results showed that much of the As in tobacco plants was As3+with little or no As5+. The presence of only inorganic As species and the absence of organic forms is evidence of lack of As methylation in plants. In assessing the amount of As species in the tobacco genotypes, it was found that ZY90contained more As3+, than FSMY. The current study highlighted that As sensitivity is not only brought about by the amount of As accumulated by a genotype but it also depends upon how and in what form the As is distributed in subcellular organs and tissues.3. The effects of phosphate on arsenic uptake and toxicity alleviation in tobacco genotypes with differing arsenic toleranceThe effects of phosphate (PO43-) on arsenate (As5+) uptake were investigated in a hydroponic culture using2tobacco(Nicotiana tabacum) genotypes (ZY90and FSMY) that differed in As5+tolerance. A total of9treatment combinations comprising As5+0,10, and100μM and PO43"-treatments0,50and500μM were used. The results showed that ZY90had greater reductions in leaf photosynthetic parameters, root and shoot dry weight, length, and nutrient content than did FSMY when exposed to As5+stress. The addition of external PO43-500μM significantly suppressed As5+(100μM) uptake in both FSMY and ZY90, with the effect being more pronounced in FSMY. Greater PO43-uptake in plants significantly reduced the influx of As5+, causing an increase in photosynthesis and nutrient uptake. Phosphate supply decreased superoxide dismutase (SOD), catalase (CAT) activity and malondialdehyde (MDA) content. The current study showed that PO43-is an effective competitive inhibitor of As5+, and it can be effectively used to control As5+accumulation in tobacco plants.