Detection, Purification and Characterization of
Cystatins from Papaya (Carica papaya) Seeds and
Leaves
Bonet, L.F.S.; Jeunon, M.F.F.; Rebello, A.N.; Siqueira-Junior, C.L.
Núcleo de Pesquisa em Biocontrole de Doenças e Proteção em Sistemas Agrícolas, Instituto de Biociências,
Universidade Federal do Estado do Rio de Janeiro
Introduction
Material and Methods
Brazil stands out as the second biggest
producer of papaya in the world, only
India’s yearly production is bigger.
But, as any other plant, papaya plants
are exposed to many biotic and abiotic stresses, which can lower growth,
production and, consequently, the
income it provides. To defend itself,
plants developed a complex defence
system, over centuries of coevolution
with herbivores, involving preformed
and latent defense pathways. In the latent, or biochemical, pathway, very important molecules are produced, such
as cysteine protease inhibitor. These
molecules interfere in insect’s digestive processes, lowering the availability of amino acids and thus lowering it’s
growt rate. It also inhibits, as the name
implies, cysteine protease activity, such
as papain, a widely used enzyme that is
produced in papaya plants’ latex. Understanding how these two chemicals
coexist in the plant body is one of the
objectives of this work, that focuses on
cysteine protease inhibitor, the phytocystatines.
The steps pictured above were executed for both the seeds and the leaves. The seeds had the sarcotesta removed by friction,
other than that they received no type of treatment. The seedlings were germinated and cultivated in a controlled environment, until they were ready to have their defences induced. This was done in two ways, mechanical wounding and exposure
to methyl jasmonate (MeJa) vapors. Protein extraction was only executed after a certain period of induction time, 24 and 48
hours, precisely.
Results and Discussion
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Papain Activity Inhibition from Leaf Extracts
100%
90%
Papain Activity Inhibition from Seed Extracts
80%
70%
60%
50%
40%
30%
20%
Leaf Control
Leaf Wounding
Leaf Systemic
Leaf MeJa
24H
93,05%
78,06%
89,54%
68,34%
48H
94,52%
91,93%
88,95%
77,63%
Figure 1: Cysteine protease inhibitor detection in papaya
leaves. The darker blue represents samples that received treatment for 24 hours, while the lighter blue represents samples
treated for 48 hours. Control, wounding, systemic and MeJa
correspond to the different induction methods. In each assay,
50µg of total protein were incubated with 1µg of papain and
BANA, the enzime’s substract. The numbers are the mean of a
series of three experiments.
10%
0%
Seed Control
Seed
Seed Boiled
Seed Precipitate
100,00%
58,70%
86,20%
37,70%
Figure 2: Cysteine protease inhibitor detection in papaya
seeds. In each assay, 50µg of total protein were incubated with
1µg of papain and BANA, the enzime’s substract. The numbers
are the mean of a series of three experiments, with the exception of the sample precipitated in ammonium sulfate.
Figure 3: Eletrophoretic analysis of papaya leaf proteins. The samples were
fractioned in SDS-PAGE 10% acrylamide. 24H and 48H correspond to the
period of induction of the seedlings, while the following correspond to the
method of induction: PM - molecular weight marker; Ctrl - control samples;
Frd - wounding samples; MJ - methyl jasmonate samples. To each lane, 50µg
of total protein were applied.
Figure 4: Immunodetection of cysteine protease inhibitors in papaya leaves. Primary antibody consisted of policlonal antibodies produced
against tomato cystatine(1:10000), while the secondary consisted of a
protein A peroxidase conjugate (1:1000). 24H and 48H correspond to
the period of induction of the seedlings, while the following correspond
to the method of induction: PM - molecular weight marker; Ctrl - control
samples; Frd - wounding samples; MJ - methyl jasmonate samples. Reactive bands are indicated by the arrows.
Figure 5: Immunodetection of
cysteine protease inhibitors in
papaya leaves. Primary antibody
consisted of policlonal antibodies produced against tomato cystatine(1:10000), while the secondary
consisted of a protein A peroxidase
conjugate (1:3000). The acronyms
correspond to the following: PM molecular weight marker; EB - protein extract; 50-90% - precipitated
preotein extract in said fraction.
Figure 6: Immunodetection of cysteine
protease inhibitors in papaya seeds. Primary antibody consisted of policlonal
antibodies produced against tomato cystatine(1:10000), while the secondary consisted of a protein A peroxidase conjugate
(1:3000). The acronyms correspond to the
following: EB - protein extract; 50-90% precipitated preotein extract in said fraction. Reactive bands are indicated by the
arrows.
Conclusion
•Papaya leaves and seeds produce phytocystatines consitutively. This production can be enhanced in the leaves
via defence induction, especially by MeJa vapors, which
resulted in the highest level of papain activity inhibition.
•In papaya leaves, two isoforms of cystatine were detected, one with ~110kDa and another with ~100kDa. In the •One of these cystatin isoforms likely has high thermostaseeds, two isoforms were also detected, one with ~66kDa bility, as protease inhibition is still detected after boiling
and the other with ~45kDa, both still appeared in the 50- the seed extract.
90% fraction of the ammonium sulfate precipitation.
Bibliography
•ANANTHAKRISHNAN, T.N. (1999) Induced responses, signal diversity and plant defense: implications in insect phytophagy. Curr. Sci. 76: 285.
•AZARKAN, M., et al. (2004) Detection of three wound-induced proteins in papaya latex. Elsevier. Phytochemistry, 64. 525:534.
•BENHAMOU, N. (1996) Elicitor-induced plant defense pathways. Trends in Plant Science. 1: 233.
•LIMA, R. C. A., et al. (2001) Etiologia e estratégias de controle de viroses do mamoeiro no Brasil. Fitopatologia Brasileira, 26:689.
•RYAN, C.A. (1990) Proteinase inhibitors in plant: Genes for improving defenses against insects and pathogens. Annual. Review of Phytopathology. 28: 425.
Bonet - [email protected]
Jeunon - [email protected]
Rebello Siqueira-Junior -
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