Meeta Jain

Effect of As on chlorophyll and protein contents and enzymic activities in greening maize tissues

Arsenic is known to have several phytotoxic effects and one of them is the reduction in chlorophyll content. Determination of chlorophyll content is often accomplished to assess the impact of most environmental stresses, as the pigment content is linked to the visual symptoms and photosynthetic plant productivity. Hence, the present investigation was undertaken to study the effect of As on chlorophyll synthesis with a view to gain some insights into the possible mechanism. Supply of 0.01 to 1.00 mM Na arsenate to the greening maize leaf segments decreased the chlorophyll content as well as chlorophyllase activity, but decline in former was more pronounced than latter at high concentration of As. Supply of arsenate also reduced total RNA, protein and acid soluble thiol content of the tissue. The inhibitory effect of As was less pronounced in the beginning i.e. upto 4 h, but became substantial with increasing time periods. Nitrate reductase activity was inhibited more strongly than chlorophyll formation and was induced only slightly by the supply of inorganic nitrogen in the presence of As. Inclusion of KNO3 had almost no effect on inhibitory effect of As on chlorophyll formation, while NH4Cl and NH4NO3 substantiated it. Percent decrease in chlorophyll content was reduced by 2-Oxoglutarate, glutamine, glycine and Na succinate, but it was increased by MgCl2 and remained unaltered by glutamate. Amongst the thio compounds tested, cysteine and 5,5′ dithio bis 2 nitro benzoic acid could overcome the toxic effect of As partially, while mercaptoethanol and dithiothreitol enhanced it and reduced glutathione had no effect. It is suggested that toxic effect of As on chlorophyll biosynthesis involves limited availability of δ-aminolevulinic acid and thiol modulation of proteins.

Inhibition of 5-amino levulinic acid dehydratase activity by arsenic in excised etiolated maize leaf segments during greening

In vivo as well as in vitro supply of sodium arsenate inhibited the 5-Amino levulinic acid dehydratase (5-aminolevulinate-hydrolyase EC 4.2.1.24, ALAD) activity in excised etiolated maize leaf segments during greening. The percent inhibition of enzyme activity by arsenate (As) was reduced by the supply of KNO3, but it was increased by the glutamine and GSH. Various inhibitors, such as, chloramphenicol, cycloheximide and LA, decreased the % inhibition of enzyme activity by As. The % inhibition of enzyme activity was also reduced by in vivo supply of DTNB. The enzyme activity was reduced substantially by in vitro inclusion of LA, both in the absence and presence of As. In vitro inclusion of DTNB and GSH inhibited the enzyme activity extracted from leaf segments treated without arsenate (-As enzyme) and caused respectively no effect and stimulatory effect on arsenate treated enzyme (+As enzyme). Increasing concentration of ALA during assay increased the activity of -As enzyme and +As enzyme to different extent, but double reciprocal plots for both the enzymes were biphasic and yielded distinct S0.5 values for the two enzymes (-As enzyme, 40 micromol/L and +As enzyme, 145 micromol/L) at lower concentration range of ALA only. It is suggested that As inhibits ALAD activity in greening maize leaf segments by affecting its thiol groups and/or binding of ALA to the enzyme.

Inhibition of Chlorophyll Synthesis and Enzymes of Nitrogen Assimilation by Selenite in Excised Maize Leaf Sefments during Greening

Supply of 0.1 mM sodium selenite (Na2SeO3 to excised maize leaf segments during greening reduced total chorophylls and inhibited in vivo nitrate reductase activity (NRA). Inhibition of chlorophyll formation by selenite was affected by ammonium but remained unaltered by nitrate. Selenite also inhibited NADH-glutamate dehydrogenase (NADH-GDH) and NADH-glutamate synthase (NADH-GOGAT) activities. Inclusion of ammonium enhanced the inhibition of NADH-GDH activity by selenite but decreased the inhibition of NADH-GOGAT activity by selenite. The results suggest the involvement of the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway of ammonia assimilation in selenite toxicity during chlorophyll formation in greening maize leaf segments.

Modulation of δ-aminolevulinic acid dehydratase activity by the sorbitol-induced osmotic stress in maize leaf segments

Osmotic stress induced with 1 M sorbitol inhibited δ-aminolevulinic acid dehydratase (ALAD) and aminolevulinic acid (ALA) synthesizing activities in etiolated maize leaf segments during greening; the ALAD activity was inhibited to a greater extent than the ALA synthesis. When the leaves were exposed to light, the ALAD activity increased for the first 8 h, followed by a decrease observed at 16 and 24 h in both sorbitol-treated and untreated leaf tissues. The maximum inhibition of the enzyme activity was observed in the leaf segments incubated with sorbitol for 4 to 8 h. Glutamate increased the ALAD activity in the in vitro enzymatic preparations obtained from the sorbitol-treated leaf segments; sorbitol inhibited the ALAD activity in the preparations from both sorbitol-treated and untreated leaves. It was suggested that sorbitol-induced osmotic stress inhibits the enzyme activity by affecting the ALAD induction during greening and regulating the ALAD steady-state level of ALAD in leaf cells. The protective effect of glutamate on ALAD in the preparations from the sorbitol-treated leaves might be due to its stimulatory effect on the enzyme.