Journal

Journal of Applied Horticulture, 10(1): 50-53, 2008

Appl

Effect of heavy manuring of phosphorous and its toxicity

on growth, photosynthesis and photosynthetic pigments in
Zn-efficient genotype of spearmint MSS-5

A. Misra and P. Singh

Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, India. E-mail: [email protected]

Abstract
Changes in growth attributes, photosynthesis (Pn), photosynthetic pigments with γ-Glu.cys peptidase peptide and Zn accumulation in
a Zn-efficient genotype of spearmint MSS-5 were investigated. Effect of phosphorus toxicity on MSS-5 were significantly different
than the other genotypes; Arka, Neera and control (the local strain), in terems of phenotypic changes in height and a decrease in
chlorophyll contents and CO2 exchange rate. Heavy P manuring lead to the tolerance of Zn accumulation in MSS-5 with γ-Glu.cys.
peptidase peptide with high protein contents and Pn. Hence, the P toxicity induced a differential utilization of γ-Glu.cys.peptidase
peptide for higher accumulation of Zn in MSS-5 spearmint with higher photosynthetic rate for increasing the height and essential
monoterpene oil(s). The study also indicated that accumulation of toxic heavy metal-Zn with γ-Glu.cys.peptidase peptide made
protein synthesis easier with antioxidants Zn cofactor enzymes.
Key words: Spearmint, Mentha spicata, Zn-efficient genotype MSS-5, protein, photosyntheis, photosynthetic pigments, Zn toxicity.

Introduction as a specific indicator of metal tolerance in plants (Grill et al.,

1988), and the role of heavy metals in decline and damage to forest
The heavy and unmanaged fertilization of major nutrients viz., N, ecosystem (Gawell et al., 1996). Zinc is potentially toxic metal
P & K for the improvement of crop yields, leads to the deficiency when transferred from plants via food chain to human. Agriculture
of micronutrients. Heavy doses of P and its precipitation to system is also the principle sources of Zn, includes the heavy P
phosphate salts makes Zn unavailable to the plant (Marschner, fertilization for crops (Misra and Sharma, 1991).
1986). Zn shows the toxicity in medicinal and aromatic plants
cultivation (Misra and Ramani, 1991). Furthermore, the acidic Possibly Zn tolerant spearmint efficient genotypes MSS-5
soil of hills bears the Fe toxicity in rose cultivation. One way cultivation in fields leads, for the removal of toxic heavy metal Zn,
or other, the toxicity of Zn and Fe especially in micronutrients from heavily phosphorous fertilized soil, for the establishments of
usage affects the productivity and secondary plant products. tolerant varieties capable of synthesizing more γ-Glu-cys peptides
The phytoremediation is a technique that plants uses to cleanup or phytochelatins. The essential monoterpene oil(s) of this efficient
contaminated soil and water. No work has been reported so genotypes is commercially important for pharmaceutical and
far on the cultivation of medicinal and aromatic plants on Fe aromatic industries. The efficient genotype MSS-5 due to toxicity
efficient genotypes to cleanup the contaminated soil with heavy of heavy metals produces the Zn induced Fe deficiency symptoms.
metal Zn. Therefore, a detailed study on the growth and physiology of
an efficient genotype MSS-5 alongwith Arka and Neera was
Spearmint (Mentha spicata L.) is widely cultivated to obtain conducted to compare Zn accumulation and tolerance against the
the essential monoterpene oil(s). Zn efficient genotype, MSS-5
phosphorous toxicity and to show the Zn induce Fe deficiency in
has been taken up for the complete exploitation of monoterpene
genotypes with primary plant products –photosynthetic pigments
oil(s), with the aim of phytoremediation and to extract out Zn
and Pn, and simultaneously the essential monoterpene oil(s)in
from Zn toxic fields. The aim to use this Zn efficient genotype
spearmints, in heavly phosphorus fertilized crops.
MSS-5 was to know the physiology and growth behaviour in
the wake of phytoremediation processes. In phytoremediation
Materials and methods
some plants tolerate high levels of toxic micronutrient metals by
a variety of mechanisms such as reduced uptake, active efflux The experiment was conducted in controlled glasshouse
and intracellular and/or extracellular sequestration. The most condition from December to March at an ambient temperature of
predominant molecules in intracellular detoxification, widely 27+3oC and with 11 h day length. Uniform suckers of spearmint
prevalent in eukaryotes are thiol tripeptides, γ-Glu-Cys peptides cultivars viz., MSS-5, Arka, Neera and a local strain (control)
or phytochelatins. These are synthesized by phytochelatin were grown in 5,000 cm3 plastic containers containing nutrient
synthase which is activated by subleathal metal concentrations solution (Hoagland and Arnon, 1950). Each 2.8 Fe μg mL-1
and play a crucial role in cytosolic metal detoxification (Steffens, treatment and mint strain were replicated 6 times and put in
1990; Zenk, 1996). The physiological concentrations of these completely randomized block design with complete 5.6 Fe μg
intracellular metal binding ligands have sometimes been used mL-1 strength, nutrient solution, were taken under the existing
 Phosphorous and its toxicity on growth, photosynthesis and photosynthetic pigments in spearmint 51

studies. The composition of nutrient solution was (as mg L-1): by adding 3.6 N HCl and precipitated protein was removed by
102 K, 100 Ca, 70 N-NO3, 16 S, 12 Mg, 9 Cl, 5 P, 0.52 B, 0.33 centrifugation. The protein was estimated by the method of
Mn, 0.33 Mo, 0.10 Zn, 0.02 Cu and Fe was as Fe-EDTA (Ferric Lowry et al. (1951). Separation of PC peptides was done on
ethylinediamine tetraacetate). During the study, instead of 5 mg a reverse phase C-18 column (μ Bondapack, RP 4 μm) with a
L-1, 10 mg L-1 phosphorous was added in nutrient solution for linear gradient 0.1% Trifluoro acetic acids at a flow rate 0.5 ml
toxicity in each treatments of 2.8 and 5.6 μg Fe mL-1. Initial pH min-1 using the applied biosystem HPLC (model No. 783A) at
of the nutrient solution was 6.7 to 6.8, which was monitored and 220 nm. Experiments were done and HPLC in triplicates, and
adjusted periodically Zn with 1.0 m M KOH or 2.0 m M H2SO4 repeated thrice.
to maintain a value of 7.2.
Pn and essential monoterpene oil(s): Initially, Pn of the
MSS-5, Arka and Neera genotypes when subjected to Fe third leaf was measured in a closed system using a poratable
deficiency stress (2.8 mg Fe mL-1 treatment), resulted in root computerized photosynthesis model Li-6000(Licor, Lincoln,
exudation, which decrease the pH of the nutrient medium and USA), as described in Singh et al.(1999) and total essential
showed the chlorosis of younger leaves whereas older one remains monoterpene oil(s) were extracted by 100 g fresh chopped leaves
green. The root exudation and their ability to absorb and utilize in Clevenger’s apparatus (Clevenger, 1928).
iron in the ferrous form vary, with the genotypes of the crop plants.
All measurements were taken in triplicate and the results
The efficient genotype of spearmint MSS-5 only turned green
are given as means±SE. The data were analyzed statistically
after the Fe deficiency visualizes characters, where as the severe
by two way ANOVA followed by ‘t-test’ for comparing the
chlorotic Arka and Neera did not turn green. Chlorosis in terms of
means following Armitage (1971). The correlation coefficient
total chlorophyll were estimated for the cultivar MSS-5 genotype
among the characters were also analyzed further the values are
which behaves as a Fe efficient genotype with more root exudation
statistically analysed by paired t-test.
which was measured with the method of Arnon (1949).
The cultivar MSS-5 genotype behaves as a Fe efficient genotype Results
with more root exudation of phenolic compounds, especially the
The efficient genotype of spearmint cv MSS-5 exudes more
caffeic acids. The phenolic compounds, the caffeic acids were
root exudates in the nutrient medium (0.54 mg g-1) followed
estimated in root exudation by the method of Singh et al. (2001).
by more acidic medium than the Arka and Neera Chlorosis of
The plan tissue Fe and Zn contents were estimated with 1 N HCl
younger leaves were more pronounced in MSS-5 with more and
extracts on atomic absorption Pye Unichem, 2900 (Misra, 1992).
more root exudates the older leaves became green, then the Arka
The lignin was estimated by the Kalson method of Browning
and Neera cultivars (Table 1). Results showed that the most Fe
(1967), 5 g samples were digested with 72% H2SO4, then diluted
efficient genotype is MSS-5, where the iron uptake is 1440 μg
with acid; to hydrolize and solubilize the polysachharides. The
g-1 in roots tissues of spearmint with further more the recovery
insoluble residue was dried and weighed as lignin on % basis.
of chlorotic younger leaves. Converting more Fe for Fe uptake in
This partially solubilized as acid soluble lignin from spearmint,
an efficient genotype by the help of maximum production of 0.82
were further quantified on UV absorbance at 410 nm.
mg g-1 phenolic compounds and root exudates -Fe+++ reductants
HPLC analysis of γ-Glu-cys peptides or phytochelatins (PCs): chelation to produce more Fe++ availability to the plant. Table
For the separation of PCs, HPLC analysis was performed in 2 indicates more toxic Zn uptake in tissue concentrations in
crude extracts of plant tissue following the method of Grill et MSS-5 cultivars (94 μg Zn g-1), with maximum production of
al. (1991). Frozen plant tissue (1 g FW) was homogenized in 0.5 protein 1.49 mg g-1 and phytochelatins 45.79 m mol g-1 FW. The
ml 1 N NaOH containing 1 mg mL-1 sodium borohydride. Alter role of Zn excess in spearmint is to behave as an antioxidant,
centrifugation at 13,000 g at 4oC, the supernatants were acidified as a scavenger to the excess free radicals removal during the
Table 1. Effects of different genotypes of spearmint in deficient Fe nutrition in younger leaves (at 2.8 μg Fe ml-1) in phosphorous excess
Genotype Chlorophyll Phenolic Caffeic acid Fe uptake Lignin P
(mg g-1) compounds (mg g-1) (μg g-1) (g g-1) value
Deficient Recovered (mg g-1)
Arka 2.01 2.07 0.65 0.41 1109 1.1 0.01
Neera 1.99 2.01 0.68 0.47 1163 1.2 NS
MSS-5 1.85 3.11 0.82 0.54 1448 1.4 0.01
Control 1.89 3.01 0.71 0.51 1437 1.1 0.01
Table 2. Effect of different genotype of spearmint in deficient Fe nutrition in younger leaves (at 2.8 μg Fe ml-1) in phosphorous excess
Genotype Height Zn Pn Protein Oil γ-Glu Cys P
(cm) accumulation (mg (CO2) (mg g-1) (%) peptide value
(mg g-1) m s )
-2 -1
(m mol g-1 FW)
Arka 31 70 124±7* 1.01 0.42 6.48±0.3 0.01
Neera 25 82 239±4 1.24 0.49 33.49±1.5 NS
MSS-5 42 94 249±2** 1.49 0.56 45.79±2.1 0.01
Control 32 90 241±3* 1.37 0.47 41.62±2.1 0.01
*, ** mean values significant at P=0.05 and P=0.01, respectively (Paired t test); NS - nonsignificant
52 Phosphorous and its toxicity on growth, photosynthesis and photosynthetic pigments in spearmint

Table 3. Correlation coefficients between different characters viz., phenolic compounds, caffeic- acids, Fe uptake, Zn accumulation in M. spicata
Characters Phenolic Caffeic Zn Fe Lignin Protein
compound acid accumulation ptake
γ-Glu.-Cys.-peptide 0.719** 0.712** 0.871** 0.641* 0.179 0.971**
Phenolic compound 0.699* 0.811** 0.821** 0.827** 0.642**
Caffeic acid 0.749** 0.714** 0.912** 0.617
Zn-accumulation -0.497 0.679* 0.747**
Fe uptake 0.579 0.621*
Lignin 0.639*
*, ** values are significant P=0.05 or P=0.01, respectively
metabolism of essential monoterpene oil(s). association showed that the phytoremediation processes remove
the toxic Zn from the heavily P fertilized fields by the Fe-efficient
Table 3 indicated the γ-Glu Cys. peptidase peptide for
MSS-5 genotype. The lignin association with Zn accumulation
significantly associated with toxic Zn-accumulation (r=0.871,
further support for increased insect resistance in spearmint
P< 0.01). Zn accumulation and lignin (r=679, P< 0.01) and Zn
accumulation with protein (r=747, P< 0.01), respectively. genotype MSS-5.

Discussion Acknowledgement
The authors are thankful to the Director, Central Institute of
Results indicated the most efficient and inefficient genotype of
Medicinal and Aromatic Plants, Lucknow for encouragement
all the existing cultivars viz., Arka, Neera and MSS-5. The most
and facilities provided.
Fe-efficient genotype is MSS-5. The root exudation of phenolic
compounds (10.87 mg g-1) and caffeic acid (0.54 mg g-1), is more
in MSS-5 cultivar where as Arka and Neera had lesser amount.
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