Seeds of Zea mays L. cv. Ganga safed-2, were surface sterilized with HgCl₂ (0.1%) for 1 - 2 min followed by thorough washing and then soaked in distilled water for half an hour. About 20 seeds were placed in petri plates lined with moistened whatman filter paper and the seedlings were grown for three days in continuous light of 30 Wm⁻² at 26˚C ± 2˚C. For some experiments, the seedlings were raised in continuous dark for three days and then used for treatment, which reflected the analysis of light induced chlorophyll biosynthesis. For Zn treatment, 4 uniformly grown seedlings were transferred to 40 ml of 0 - 5 mM ZnCl₂ solution contained in small petri plates for 24 h in continuous light as above and the treated seedlings were used for various analyses.

Determination of growth and Zn content―Treated seedlings were used to analyze Zn content, fresh weight, dry weight, number of roots, weight and length of root and shoot. For Zn content analysis, root and shoot tissue of the seedlings were dried in oven at 80˚C for 72 hours. The dried material (400 mg) was wet digested with 5 ml of conc. HNO₃ and diluted 5× to 50× times i.e. (5× (control), 10× (0.001, 0.01, 0.1 mM), 20× (1 and 2 mM) and 50× (5 mM) with distil water. Zn content was determined by using atomic absorption spectrophotometer, make “GBC, Scientific Equipment Ltd―AVANTA”.

Biochemical parameters―Total protein and RNA content of root and shoot tissue were measured by the method of [12] and [13] , respectively. For protein content analysis, treated tissues were first boiled with and then extracted using 5.0 ml of 80% ethanol for each step. The extract was centrifuged at 5000 rpm for 10 min and the pellet was suspended in 5.0 ml of 10% TCA for 30 min and again centrifuged similarly. The resulting pellet was dissolved in 5.0 ml of 0.1 N NaOH and kept for at least 1hr. After centrifugation, the supernatant was used for protein estimation with Folin Ciocaulteau’s phenol reagent. For RNA extraction similar 80% ethanol treatment was used and then the pellet was suspended in 2.0 ml of 1% PCA mixed thoroughly and centrifuged. The pellet was treated with 5.0 ml of ethanol: diethyl ether: chloroform (2:2:1 v/v) mixture and then centrifuged. Three ml of 0.3 N KOH was added to the residue and was incubated at 37˚C for 18 hr. Next the pH was adjusted to 2.0 using 1 N PCA, centrifuged and appropriately diluted supernatent was used to estimate total RNA by orcinol reagent. For proline content determination, method of [14] was used.

Chlorophyll extraction and estimation―Pigments were extracted from the treated shoot tissue using 80% acetone and estimated spectrophotometrically by measuring the absorbance at 470 nm, 646 nm, and 663 nm. The pigment content was calculated using the following equations according to the method of [15] .

Chla ( μgml − 1 ) = 12 . 21 ( A 663 ) − 2 . 81 ( A 646 )

Chlb ( μgml − 1 ) = 2 0. 13 ( A 646 ) − 5 .0 3 ( A 663 )

TotalChlorophyll = Chla + Chlb

Caratenoids ( μgml − 1 ) = 1 000 ( A 47 0 ) − 3 . 27 ( Chla ) − 1 0 4 ( Chlb ) / 229 .

ALA content determination―Estimation was performed according to the method of [16] . For extraction of ALA, shoot tissue was extracted with 1 M sodium acetate buffer. Homogenate was further centrifuged at 10,000 rpm for 10 min at 4˚C. ALA was reacted with ethyl acetoacetate followed by modified Ehlrich reagent for estimation. Color formation was measured at absorbance 555 nm on Shimadzu UV-1800 spectrophotometer.

ALA Formation―method of [16] was used to estimate the activity. Light and dark grown seedlings were used which were further incubated with levulinic acid which is responsible for inhibition of ALA synthesis. Incubation was performed in light and dark conditions. Modified Ehlrich reagent was used to estimate amount of ALA as above.

ALAD assay―ALAD activity was assayed spectrophometrically by estimating the amount of PBG (porphobilinogen) formed using modified Ehlrich reagent [17] according to the method of [18] . Treated shoot was extracted in 50 mM Tris HCl buffer (pH 8.2) containing 10 mM DTT. Extract was centrifuged at 15,000 rpm for 30 min at 4˚C and supernatant was used as enzyme preparation. Reaction mixture of ALA, enzyme and MgCl₂ was used to which TCA was added to stop the reaction. One unit of enzyme activity is defined as 1 nmol of PBG formed per hour.

Data presented in the paper are average of at least four independent experiments with ± S.E. Significance of difference obtained for various treatments was tested by the Student’s t-test. Compound correlation was calculated by Graphpad Prism 7.04.

Supply of 0 - 5 mM ZnCl₂ to three days old maize seedlings for 24 h resulted in concentration dependent increase in accumulation of Zn in root as well as shoot tissue with the effect being more substantial in the former (Figure 1). Sharp increase in Zn content in root tissue was observed at/above 0.1 mM, while in shoot

tissue the increase was gradual; hence, perfect correlation resulted for later with R² values being 0.591 and 0.976, respectively (Figure 1).

Zinc treatment of maize seedlings reduced significantly the fresh wt of the seedlings at higher concentrations of 1 - 5 mM, while the dry wt was reduced slightly (Table 1). Root parameters, like number and length were decreased more prominently and significantly in the concentration range of 0.1 to 5 mM Zn, but effect on shoot length was less severe and less significant (Table 1). Further, root wt was decreased more prominently and significantly than shoot weight. Moreover, all the growth parameters were correlated with Zn treatment with R² values being in the range of 0.542 (For root length) to 0.744 (For shoot wt).

Incubation of maize seedlings with ZnCl₂ increased the protein content in both the root as well as shoot tissue with the effect being more prominent and significant in former (Table 2). Proline content was also increased in both the tissue with more significant change in roots (Table 2). The RNA content in shoot tissue was decreased, while it was increased in root issue but the effect was not significant except at 1 mM Zn (Table 2).

Compound correlation analyses yielded correlation between each pair of the parameters. Thus, in root tissue a perfect correlation of Zn content with root wt and protein and also between root wt and protein was observed having the values −0.989, 0.947 and −0.969, respectively (Table 3(a)). For Zn content, root wt and protein with RNA and proline, strong correlation with values being in the range of 0.654 to 0.740 resulted, however between RNA and proline there was no correlation (Table 3(a)). In shoot tissue perfect correlation was observed for Zn content and shoots wt with proline having the values 0.939 and −0.949, respectively (Table 3(b)). Very strong correlation of shoot wt with Zn content

Values relative to control are given in parentheses. The level of significance tested was denoted as p values * < 0.05, ** < 0.01, *** < 0.001.

Values relative to control are given in parentheses. The level of significance tested was denoted as p values * < 0.05, ** < 0.01, *** < 0.001.

(−0.854) and RNA (0.826), strong correlation of protein with Zn content (0.632), shoot wt (−0.787) and proline (0.727) and between RNA and proline −0.670) also resulted (Table 3(b)). While for RNA with Zn content and protein lesser correlation was observed.

Treatment of light grown maize seedlings with ZnCl₂ at 1 - 5 mM increased the chlorophyll, carotenoid and ALA content with significant effect for carotenoids at 5 mM Zn and for ALA at 2 and 5 mM both (Table 4(a)). To analyse ALA formation activity ZnCl₂ treated seedlings were incubated with 60 mM levulinic acid for 4 h either in light or dark corresponding to chloroplastic and mitochondrial activities respectively, the ALA accumulation in both was slightly enhanced and exerted no correlation (Table 4(b)), However, ZnCl₂ significantly increased ALAD activity at all the concentrations (Table 4(b)).

The compound correlation analysed for chlorophyll and carotenoid contents with Zn content exhibited very strong +ve correlation (0.862) but had shown very strong −ve correlation (−0.836 and −0.807) with shoot wt (Table 5(a)). For ALA with Zn content the correlation was strong being 0.703 and with shoot wt it was

perfect being −0.908 (Table 5(a)). Further, observed correlation of chlorophyll with carotenoid and ALA was perfect being 0.992 and 0.891, respectively and between carotenoid and ALA was 0.870 (Table 5(a)). Positive correlation for Zn content with ALA formation in light and dark was lesser (0.527 and 0.590), but for ALAD activity was very strong (0.875). Observed correlation of shoot wt with these parameters was negative and stronger being perfect with ALAD (−0.979) (Table 5(b)). Further, ALAD activity exerted strong correlation with ALA formation in light and dark both being 0.658 and 0.852, respectively and stronger with later (Table 5(b)).

Treatment of dark grown maize seedlings with ZnCl₂ decreased the chlorophyll content slightly and the carotenoid content remained unaffected, while the ALA content was significantly increased at 2 and 5 mM ZnCl₂ (Table 6(a)). The observed correlation with Zn treatment was found to be very strong for ALA content with R² value of 0.818 (Table 6(a)). When maize seedlings were treated with ZnCl₂, ALA formation activity both in light and dark and ALAD activity were enhanced at 1 and 5 mM showing significant increase for ALA formation in light at 1 mM treatment only (Table 6(b)). Zn treatment and these parameters also exerted strong correlation with R² value of 0.960, 0.728 and 0.691 respectively (Table 6(b)).

Zn is an essential micronutrient for plants, but is toxic at high concentrations, hence its effect on growth depends upon the concentration. Thus decrease in growth due to Zn supply has been reported in ryegrass [5] , sugar beet [7] cluster bean [6] and Sorgham [8] , however, in mungbean [19] and Eruca sativa [9] increased growth resulted. In the present study, with the supply of ZnCl₂ to 3 days old maize seedlings, the effect on Zn accumulation and morphological and biochemical growth parameters have been analysed. The Zn treatment led to gradually increased accumulation of Zn in the shoot tissue, while in the root tissue substantial increase was observed at/and above 0.1 mM ZnCl₂ (Figure 1). Also the root parameters were significantly reduced at/and above 0.1 mM ZnCl₂ (Table 1). Thus Zn supply significantly reduced the overall growth of maize seedlings mostly at 1 - 5 mM ZnCl₂ exerting strong correlation and the observed effect was more substantial for root tissue. Amongst biochemical parameters, increase in protein and proline content was more prominent in root tissue than the shoot, while RNA content was reduced in shoot tissue (Table 2). Reduction in RNA may be due to faster degradation and the increased protein due to translation of pre synthesized RNA. It seems that enhanced protein and proline synthesis is associated with Zn effects in root, as these contents are significantly increased. Increased levels of protein may be required for the synthesis of stress induced proteins [20] and of proline, for osmotic adjustments in response to Zn. Accumulation of proline is well documented against various stresses in plants, such as, Spinacia oleracea [21] and Cucurbeta pepo [22] . Further, the observed correlation for Zn accumulation in root with as well as between root wt and protein was perfect (Table 3(a)). Also perfect correlation for Zn accumulation in shoot and shoot wt with proline resulted (Table 3(b)). Thus the Zn accumulation and growth effect in maize depends upon the tissue with root as the target site.

Chlorophylls, the vital photosynthetic pigments and carotenoids, the light harvesting pigments in green tissues affect the overall photosynthetic activity and hence the growth and development of the plant. Chlorosis due to both Zn deficiency [23] and toxicity [2] have been observed in plants. Reports of decrease in chlorophyll content due to Zn supply in sugar beet [7] , cluster bean [6] and Sorgham [8] but increase in mungbean [19] has been documented. In the present study, Zn treatment to light grown seedlings prominently increased the chlorophyll and carotenoid content, while in dark grown seedlings it had marginal/no effect (Table 4(a) and Table 6(a)). This indicated that the steady state level of the pigments is getting affected by Zn rather than inducible level. Content of ALA, a regulatory metabolite and universal precursor for all the tetrapyrroles, was increased at higher Zn concentrations in both the regimes (Table 4(a) and Table 6(a)). Thus increased synthesis of chlorophylls as well as other heme containing proteins is likely to result by Zn supply. Increased ALA content may involve enhanced synthesis and the ALA synthesis was also found to be increased in both the regimes in light as well as dark that corresponds to chloroplastic and mitochondrial activities respectively, however, the effect was non significant (Table 4(b) and Table 6(b)). Thus it seems that Zn exerts lesser effect to enhance ALA synthesis. Further, Zn supply increased ALAD activity of light as well as dark grown seedlings being significant in former (Table 4(b) and Table 6(b)). Hence, the next step of chlorophyll biosynthetic pathway catalysed by ALAD involving formation of porphobilinogen from ALA seems to get affected by Zn. Further, ALADs from different sources are metalloenzymes that utilize a variety of divalent and monovalent cations [11] ALAD requires Mg²⁺ and Zn²⁺ for its activity [10] hence its activity is enhanced by Zn treatment. Moreover, in light grown seedlings the observed correlation for pigment content with Zn accumulation and shoot wt was very strong for both, +ve (0.862 and 0.862) and ?ve (0.836 and 0.807), respectively (Table 5(a)). Also for ALA content and ALAD with shoot wt perfect ?ve correlation resulted (Table 5(a) and Table 5(b)). Moreover, ALAD has been reported to be a substrate-modulated enzyme [24] . Hence, it is likely that during Zn treatment shoot growth and chlorophyll formation is mainly influenced by ALA and subsequently by ALAD in maize seedlings.