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Efficient oleoresin biomass production in pines using low costmetal containing stimulant paste

Kelly C. da Silva Rodrigues a, Mıriam A. Apel b, Amelia T. Henriques b,Arthur G. Fett-Neto a,*aCentro de Biotecnologia, UFRGS (Programa de Pos-Graduacao em Biologia Celular e Molecular), C.P. 15005, CEP 91501-970, Porto Alegre,

RS, Brazilb Faculdade de Farmacia, UFRGS, Av. Ipiranga 2752, 90610-000, Porto Alegre, RS, Brazil

a r t i c l e i n f o

Article history:

Received 15 May 2010

Received in revised form

22 August 2011

Accepted 24 August 2011

Available online 14 September 2011

Keywords:

Oleoresin

Pinus elliottii

Slash pine

Metal

Terpene

* Corresponding author. Tel.: þ55 51 3308 76E-mail address: fettneto@cbiot.ufrgs.br (A

0961-9534/$ e see front matter ª 2011 Elsevdoi:10.1016/j.biombioe.2011.08.021

a b s t r a c t

Oleoresin biomass production by trees of Pinus elliottii, which is a source of terpenes for the

chemical and pharmaceutical industries, was investigated. Trees were individually

analyzed for oleoresin yield using the bark streak method of wounding for stimulation of

resin flow. Controls included plain wounding and wounding followed by application of

commercial stimulant paste, based on sulfuric acid and synthetic precursors of the

phytohormone ethylene. Metal cofactors of terpene synthases and ethylene receptors

applied locally as adjuvants of the oleoresin stimulant paste on wounded bark tissue

improved resin yields. The use of potassium, copper, and iron did not affect significantly

the composition of semiochemical monoterpenes, which are involved with pine - bark

beetle interactions, and are important as oleoresin-derived products. Some adjuvants,

particularly potassium, were capable of supporting oleoresin yields equivalent to those

obtained with current commercial stimulant paste, even with removal of ethylene

precursor, the most expensive adjuvant.

ª 2011 Elsevier Ltd. All rights reserved.

1. Introduction C15) and rosin (diterpene, C20) fractions [4,5]. The potential of

The increasing need for wood, cellulose pulp, natural chem-

icals, fiber, and bioenergy has prompted efforts in developing

high yielding forest plantations, as well as a search for

alternative plant sources for these products [1e3]. Non-

timber forests products have also been drawing consider-

able attention, including as a means to replace fossil-fuel-

derived materials [4]. In this scenario, pine oleoresin seems

to be a copious, readily available, and profitable biomass

alternative, which can be extracted for several years

throughout tree life.

Pine oleoresin is made of a complex mixture of terpenoids,

consisting of turpentine (monoterpene, C10, and sesquiterpene,

42; fax: þ55 51 3308 7309..G. Fett-Neto).ier Ltd. All rights reserve

hydrocarbons and turpentine derived from oleoresin as biofuel

is also relevant [4], as exemplified by the development of effi-

cient methods of obtaining very high density biofuels from

selective dimerization of pinenes [6], major components of

pine oleoresin. Tapping pine trees to obtain terpene biomass is

an activity well suited to countries with a standing resource of

pines, leading to many economic and social benefits [7]. Brazil

has extensive areas of tree plantations (around 7 million ha),

which, in addition towood products, yield resin, rosin and gum

turpentine [8]. The national resin yield in 2009 was approxi-

mately 83 400 t, and the average price was 470.22 $ t�1 [9].

The pine rosins are abundant natural chemicals that have

many industrial applications including synthetic rubber,

d.

b i om a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 4 4 4 2e4 4 4 8 4443

coatings, paper sizing, polymerization emulsifiers, adhesive

tackifiers, printing ink resins, and waterproofingmaterial [10].

The pine turpentine has been traditionally employed as

a solvent or cleaning agent for paints and varnishes or in the

pharmaceutical industry [11]. Turpentine can be fractionally

distilled to provide its major constituents, a- and b-pinene in

pure form. Alpha and b-pinene are an important source of

intermediates and ingredients for flavors and fragrances [12].

This industry consumes approximately 30 kt y�1 of pinenes,

which are used to produce a diverse range of products [13,14].

Commercial resin tapping is normally carried out on the

trunk of fully-grown trees. The bark is mechanically removed

exposing the under-wood surface. In the “bark streak” system

[10], 2.5 cm wide strips of pine bark are removed from about

one-third of the diameter of the tree. The area fromwhich bark

is removed during the production season is called a face [15].

Resin extrudes from resin channels and drips into an open

plastic bag, placed on the wound base and attached to the tree

by ametal wire (Fig. 1). To increase resin flux and to extend the

extruding period, a chemically active paste containing sulfuric

acid is applied to the exposed fresh surface. The extrusion of

resin peaks immediately after the cutting, decreasing with

time. New bark removals are made in the trunk in an area

immediately above the previous cutting, at intervals of 15 days

[16]. Since the 1980s, 2-chloroethylphosphonic acid (CEPA) (CAS

No. 82375-49-3), an ethylene-releasing compound [17], is also

commonly present in the commercial stimulant paste [15,18].

In temperate zones, this activity is seasonal and extends from

spring to autumn, the growing seasons [12,13]; however, the

mild winter in southern Brazil can be a productive season,

Fig. 1 e Resin collection system.

improving yearly yields by approximately 20% in relation to the

total oleoresin biomass produced from spring to autumn [19].

Resin terpenes are derived from isopentenyl diphosphate

(IPP) produced by the mevalonate pathway in the cytosol-

endoplasmic reticulum or by the deoxi-xylulose-5-phosphate

pathway in the plastids [5]. The enzymes relevant to resin

terpene biosynthesis include monoterpene synthases, sesqui-

terpene synthases and diterpene synthases. Although these

enzymes lead to a variety of different products, some of their

biochemical properties are shared due to a partial conservation

of catalytic mechanisms. All three enzyme classes require

divalent cations for catalysis. Pine monoterpene synthases

require divalent cations (Mgþ2, Mnþ2, or Feþ2) and activity is

improved by Kþ [20].

Members of the genus Pinus constitutively produce and

store copious amounts of resin [5]. Pine resin production from

species growing in tropical and subtropical regions has been

less studied than those from temperate regions [21]. In the

former regions, however, bark beetles, that cause massive

perforation of the bark and vector pathogenic fungi, are not

a major threat as in North America [5].

A large field experimental study on oleoresin tapping yields

of southern Brazil was carried out with approximately 3000

28-year old Pinus elliottii var. elliottii (slash pine) trees. This

study aimed at identifying chemical adjuvants, based on

terpene synthases and ethylene receptor metal cofactors, for

reducing costs and improving the stimulant paste commer-

cially used in the tapping practice. Effects of the use of these

adjuvants in stimulant pastes were also examined in relation

to the composition of major monoterpene components of the

resin, not only because of their value for the chemical

industry, but also due to their importance as semiochemicals

in the interaction with bark beetles where these occur.

Experiments were carried out in a totally randomized

layout, using trees of internal areas of pine plantations to

avoid forest border effects. This layout was chosen on the

basis of homogeneity of trees (similar size, age and genetic

origin) and soil properties within forest sites. A total of 40e50

randomly selected trees were used per treatment, and, to

further ensure reproducibility of results, experiments were

independently replicated in two different sites (totalizing 3320

trees).

2. Materials and methods

2.1. Plant material and resin tapping operation

Resin tappingwas done by the “bark streak” system in 28 year-

old slash pine (P. elliottii Engelm. var. elliottii) trees, grown in

Rio Grande do Sul (southernmost state of Brazil), city of Sao

Jose do Norte (approximately 32� of south latitude and 52� of

west longitude) [21]. All forests used in the study had not been

tapped for resin prior to the experiments. Stimulant pastes

produced with different chemical adjuvants (Table 1) were

applied on the wounded tissue. Every experiment had two

control treatments: a negative control consisting of bark

streak without paste application, and a positive control using

the current commercially exploited stimulant paste (a

mixture of pyrogenic silica, emulsifier, and active ingredients

Table 1 e Chemical adjuvants in resin stimulating paste, concentrations examined and rationale for selection.

Chemical adjuvant Concentration Activity

Copper sulfate (CuSO4) 1 mol m�3e10 mol m�3 and 100 mol m�3 Cu: ethylene receptor cofactor [26]

Manganese sulfate (MnSO4) 1 mol m�3e10 mol m�3 and 100 mol m�3 Mn2þ: monoterpene synthase cofactor [25]

Ferrous sulfate (FeSO4$H4O) 0.5 mol m�3e10 mol m�3 and 100 mol m�3 Fe2þ: monoterpene synthase cofactor [25]

Potassium sulfate (K2SO4) 50 mol m�3e100 mol m�3 and 500 mol m�3 Kþ: monoterpene synthase activator [25]

b i om a s s an d b i o e n e r g y 3 5 ( 2 0 1 1 ) 4 4 4 2e4 4 4 84444

sulfuric acid and CEPAwith a volume fraction of 20% and 4.5%,

respectively). In order to avoid relevant differences in irradi-

ance and wind effects, in all of the experiments treatments

consisted of randomly selected trees in the inner portion of

the forest, without inclusion of the three to fourmost external

lines and rows of individuals at the forest borders. The

chemical stimulants were used isolated and/or combined.

Metal cofactors (Table 1) were tested added to the complete

commercial paste and, in some cases, in pastes without CEPA.

These conditions are described in the legends of each table of

the results. In the first year (2002e2003), 50 trees were

analyzed for each treatment (site B). In the following year

(2004e2005), 40 trees were tested for each one of selected

treatments, at a new site (site C). After the completion of the

seasonal resin-tapping period, plastic bags with resin were

collected and weighed on a field digital balance after careful

draining of rainwater. Plastic bagswere regularly checked and

replaced as required to avoid any oleoresin loss. A total of 3320

trees were treated and monitored. Trees used in the experi-

ments were within the diameter at breast height range from

20.5 to 23.5 cm. The experiments were carried out at AMBAR

FLORESTAL LTDA. forest installations.

2.2. Resin analysis

Resin samples were taken from the trunks of 90 trees at site B.

Each sample consisted of a mixture of equal proportions of

freshly flowing resin (obtained immediately after the

wounding process) from 10 individual trees submitted to the

same treatment. The samples were collected in the spring of

2003, all in the afternoon. Immediately after harvest, all

samples were frozen in liquid nitrogen and kept as such until

storage in an ultra-freezer (�80 �C) in completely sealed vials

before use. Terpene samples preparation was performed as

previously described [22]. Extractions and analyses were done

in triplicate.

Quantitative GC analysis was carried out using a GC-

PerkinElmer Autosystem XL chromatograph equipped with

TotalChrom� Workstation Software, using a DB-1 silica

capillary column (25 m � 0.25 mm). Injector and detector

temperatures were set at 220 �C and 250 �C, respectively. Theoven temperature was programmed from 60 �C (3 min) to

300 �C at 15 �C min�1. Nitrogen was employed as carrier gas

(1.3 cm3 min�1) and the injected volume was 3 mm3. The

percentage compositions were obtained from electronic

integration measurements using flame ionization detection

(FID) without taking into account relative response factors. For

quantification of a and b-pinene in resin, standard curves

were generated using authentic standard monoterpenes

(Sigma, USA).

Qualitative analyses were performed using a Shimadzu

GC-17A chromatograph coupledwith a quadrupoleMS system

(QP 5000) equipped with a DB-5 fused silica capillary column

(30 m � 0.25 mm � 0.25 mm, J & W Scientific). Helium was the

carrier gas, and the flow rate, 1 cm3 min�1. The MS operating

parameters were: ionization voltage ¼ 70 eV; ion source

temperature ¼ 250 �C. Injector and detector temperatures

were set at 220 �C and 250 �C, respectively. The oven

temperature was programmed from 60 �C to 300 �C at

3 �C min�1. For quiral analysis, a cyclodex-B column coated

with b-cyclodextrin (30 m � 0.252 mm � 0.25 mm; J&W Scien-

tific) was used. For this column, the oven temperature was

programmed from 60 �C to 200 �C at 3 �C min�1; injector and

detector temperatures were set at 220 �C and 230 �C, respec-tively. Compound identification was based on comparison of

retention indices (determined relatively to the retention times

of a series of n-alkanes) and mass spectra with those of

authentic samples and with literature data [23].

2.3. Statistical analyses

Experimental layout was totally randomized. Each treatment

had 40 to 50 replicates. Experiments were carried out in two

different sites. Simple analyses of variance (ANOVA) followed

by Tukey test were used for data evaluation. Data were

transformed when necessary to fit the ANOVA requirements

of variation homogeneity. In a case when variance homoge-

neity was not obtained (quantification of one of the terpenes

in resin analyses, m-chavicol), Welch ANOVA was used fol-

lowed by Dunnett C test. P � 0.05 was used in all tests [24].

3. Results and discussion

Similar physiologic responses were observed for trees treated

with related groups of paste constituents. Some of the metal

adjuvants tested in combination with CEPA in the first

experimental year had an important impact in resin produc-

tion when compared with the respective controls. The pastes

supplemented with Fe2þ or Kþ yielded relatively higher

contents of resin (Table 2). In most cases, the best concen-

tration of K was 500 mol m�3, whereas for Fe this value was

100 mol m�3 or 10 mol m�3. Stimulatory trends on resin

production with copper supplementation in the paste were

also noticed, particularly at lower concentrations (Table 2).

The metal adjuvants that showed better resin yield stim-

ulation capacity at site B were selected for further tests at

a new site, both isolated and combined. Trees grown at site C

produced more resin that those at the B site; however, the

sites were sampled in different years. A higher difference

Table 2 e Bioresin production at site B with stimulantpaste supplementedwith differentmetals. Numbers aftermetal symbols correspond to concentration in paste(mol mL3). Paste [ commercial formulation, barkstreak [ plain wound. Bars sharing a letter within eachmetal adjuvant and respective control treatments do notdiffer by Tukey test (P £ 0.05).

Metal [mol m�3] kg � SE

Mn [1] 3.001 � 0.128a

Mn [10] 2.861 � 0.1245a

Mn [100] 2.680 � 0.1216a

Bark streak 2.133 � 0.0732b

Paste 2.861 � 0.1292a

Fe [0.5] 3.037 � 0.1140ab

Fe [10] 3.039 � 0.1246ab

Fe [100] 3.196 � 0.1299a

Bark streak 2.133 � 0.0732c

Paste 2.861 � 0.1292b

K [50] 3.031 � 0.1379ab

K [100] 2.849 � 0.1358b

K [500] 3.468 � 0.1642a

Bark streak 2.133 � 0.0732c

Paste 2.861 � 0.1292b

Cu [1] 3.050 � 0.1146a

Cu [10] 2.880 � 0.1317a

Cu [100] 2.660 � 0.1274a

Bark streak 2.133 � 0.0732b

Paste 2.861 � 0.1292a

Table 3 e Bioresin production at site C with stimulantpaste supplementedwith differentmetals. Numbers aftermetal symbols correspond to concentration in paste(mol mL3). Paste [ commercial formulation, barkstreak [ plain wound. Bars sharing a letter within eachmetal adjuvant and respective control treatments do notdiffer by Tukey test (P £ 0.05).

Metal [mol m�3] kg � SE

Mn [1] 5.612 � 0.1284a

Bark streak 2.543 � 0.0864b

Paste 5.354 � 0.1810a

Fe [10] 6.038 � 0.1459a

Fe [100] 5.463 � 0.1168b

Bark streak 2.543 � 0.0864c

Paste 5.354 � 0.1810b

K [50] 5.706 � 0.1020ab

K [500] 5.969 � 0.1348a

Bark streak 2.543 � 0.0864c

Paste 5.354 � 0.1810b

Cu [1] 5.663 � 0.1732ab

Cu [10] 5.936 � 0.1527a

Bark streak 2.543 � 0.0864c

Paste 5.354 � 0.1810b

Table 4 e Bioresin production at site C with stimulantpaste supplemented with metal adjuvants without CEPA(*). Numbers after metal symbols correspond toconcentration in paste (mol mL3). Paste [ commercialformulation, bark streak [ plain wound. Bars sharinga letter do not differ by Tukey test (P £ 0.05).

Metal [mol m�3] kg � SE

Fe [100]* 4.341 � 0.0902a

Bark streak 2.036 � 0.0633b

Paste 4.723 � 0.1416a

K [500]* 4.785 � 0.0695A

Bark streak 2.036 � 0.0633B

Paste 4.723 � 0.1416A

b i om a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 4 4 4 2e4 4 4 8 4445

between trees treated with bark streak and paste in the

2004e2005 year at site C compared to the same treatments in

the 2002e2003 year was evident (Tables 2 and 3).

At site C, trees treated with pastes individually supple-

mented with manganese, iron, potassium and copper had

equivalent or higher resin yields compared to those of trees

exposed to the basal paste. Trees under the treatment of basal

paste supplemented with 10 mol m�3 Fe, 500 mol m�3 K, or

10 mol m�3 Cu produced higher resin quantities than their

counterparts treated with basal paste only (Table 3).

The overall results of combinedmetal adjuvants with CEPA

containing paste showed that these were effective in inducing

resin production, and all paste containing treatments (basal

and/or supplemented) improved resin yield compared to bark

streak. Trees submitted to most of the metal combinations

showed oleoresin yield statistically similar to those treated

with control paste; however, the results did not show

a consistent synergistic effect of combined metal adjuvants

on resin yield above those observed with individual metals.

Moreover, several metals combined in the absence of CEPA (K

[500 mol m�3]Fe[100 mol m�3]Mn[10 mol m�3] and K

[500 mol m�3]Fe[100 mol m�3]Mn[1 mol m�3]Cu[1 mol m�3])

resulted in yield reduction compared to the basal paste

treatment and to the yield of individual metals with CEPA

(data not shown).

The effectiveness of individual metal adjuvants supple-

mented to the paste devoid of CEPA was investigated in

additional experiments at site C. Resin yield of trees treated

with the individual metal ions replacing CEPA was equivalent

to the basal paste with CEPA (Table 4).

The analysis of monoterpene constituents of the oleoresin

derived from the various pastes at site B showed that themain

components were b-pinene and a-pinene (overall average in %

of total turpentine terpenes � sd: b-pinene ¼ 51.11 � 4.19;

a-pinene ¼ 40.74 � 4.04). b-phellandrene, camphene, M-chavi-

col andmyrcenewere theother components detected (Table 5).

The proportion of a and b-pinene in the turpentine was not

significantly affected by the metal adjuvants, the same

applying to b-phellandrene, M-chavicol and myrcene. The

proportion of camphene increased in the oleoresin of trees

treatedwithmetal adjuvant supplementedpaste, ranging from

approximately 0.5% of total turpentine terpenes in the controls

to close to 2% of total turpentine terpenes in the oleoresin of

trees exposed to metal supplemented pastes (Table 1). The

actual concentration of a and b-pinene in the oleoresin was

highly variable and did not show significant differences

between the oleoresin of control trees and metal supple-

mented, although a trend toward reduction of pinene concen-

trationwas observed in the latter (overall average and standard

Table 5 e Concentration (as % of total terpenes in turpentine) of terpenes in Pinus elliottii var. elliottii turpentine. Numbers inthe first column after metal symbols indicate metal concentration in stimulant paste (mol mL3). All treatments wereapplied to trees of site B and contained CEPA. Paste [ commercial formulation, bark streak [ plain wounding. Numberswithin the same column sharing a letter do not differ by a Tukey or (*) Dunnett C test (P £ 0.05).

Treatment a-pinene b-pinene Camphene Myrcene b-phellandrene m-chavicol (*)

Mn [1] 41.637a 50.718a 1.143abcd 0.838a 4.387a 1.052a

Fe [10] 41.843a 50.225a 1.363ab 0.707a 4.323a 0.670a

Fe [100] 38.790a 53.248a 1.298abc 0.990a 3.402a 0.573a

K [50] 37.835a 51.682a 0.978bcd 1.643a 3.168a 0.617a

K [500] 43.268a 46.457a 1.670ab 1.915a 4.725a 1.155a

Cu [1] 42.327a 50.033a 1.838a 0.713a 3.555a 0.923a

Cu [10] 36.795a 55.798a 1.672ab 0.543a 4.147a 0.445a

Paste 43.311a 52.118a 0.485d 0.623a 2.672a 0.378a

Bark streak 40.825a 49.723a 0.552cd 0.910a 5.265a 1.422a

b i om a s s an d b i o e n e r g y 3 5 ( 2 0 1 1 ) 4 4 4 2e4 4 4 84446

deviation mg g�1) e control a-pinene ¼ 63.84 � 5.24, metal

supplemented ¼ 47.11 � 7.9, control b-pinene ¼ 81.57 � 7.69,

metal supplemented ¼ 61.62 � 14.92. The higher content of

camphene in the oleoresin of trees treated with metal adju-

vants may explain the observed trend toward lower pinene

content. An evaluation of oleoresin composition from trees

treated with metal adjuvant containing paste, but devoid of

CEPA, compared to that of trees treated with commercial paste

(i.e. containing CEPA) also showed no significant differences in

a and b-pinene composition (data not shown).

In light of these results, an expansion of the use of

improved stimulant paste without CEPA, but containing K,

was carried out in comparison to commercial stimulant paste

containing the ethylene precursor. The expansion involved

monitoring the oleoresin yields of 1100 trees from the winter

of 2005 to the summer of 2008. The trees were distributed in

different sites with different characteristics, notably drainage,

being one well drained and the other subject to periodic

flooding. The overall results of year average oleoresin

production per tree were not statistically different (biomass:

kg � se): commercial paste - containing CEPA ¼ (6.741 � 1.290)

kg improved paste e devoid of CEPA, but containing

500 mol m�3 of potassium ¼ (6.867 � 1.106) kg.

The stimulatory effect of metal ions added to the paste on

the resin yield may be related to the fact that terpene syn-

thases require divalent metal cations for catalysis, and the

monoterpene synthases isolated from conifers, besides

requiring Mnþ2, Mgþ2 or Feþ2, are also stimulated by mono-

valent cations, particularly Kþ [20,25], suggesting that the

initial choice of adjuvants was appropriate. Effects of the

metal ions on the promotion of oxidative stress are possible,

but can hardly be regarded as significant for the resin yield,

given the extensive wounding applied to the trunk and the

presence of relatively high concentration of sulfuric acid in

the basal paste composition of the treatments. In addition,

stimulatory effects were not always proportional to the

concentration of metal adjuvant and combinedmetals had no

consistent stimulatory effect; this evidence is not supportive

of a metal-induced oxidative stress response as the main

cause of oleoresin increase. The positive effects of copper

supplemented paste on resin yield could be related to its

action as a cofactor of the ethylene receptor (ETR1) [26], since

ethylene has been closely associated with resin production

upon wounding in coniferous species [10,19,21,27e32]. The

limited effect of manganese containing paste without CEPA

may indicate that there are sufficient amounts of this enzyme

cofactor in the wounded vascular tissue to support overall

oleoresin yield.

Trees grown at site C producedmore resin that those at the

site B; however, one must be careful in comparing this result

since recorded yields in the former were in 2004e2005,

whereas for the latter sampling years spanned 2002e2003.

Official climate records for the region [33] revealed that the

spring of 2002 e summer 2003 were rainy and the winter of

2003, mild. On the contrary, spring 2004-summer 2005 were

very dry (driest of the previous 10 years) and the winter of

2005, slightly colder than usual. The drought period of

2004e2005 may have improved the yields of resin by the trees

and prompted tree response to applied pastes. Exposure to dry

periods has been shown to promote terpene production in

other pine species, presumably by reducing growth and

allowing higher carbon and nutrient allocation to secondary

metabolism as a defense strategy [34]. Moreover, droughtmay

also induce oxidative stress, thereby activating defense

responses based on secondary metabolites [35]. Previous

studies comparing paste-stimulated resin yield of various

tropical pine species with that of P. elliottii grown in the

southeast of Brazil showed that production was negatively

affected by low temperatures, particularly that of P. elliottii

[36]. The results from the current study also showed that the

basal paste and the majority of the modifications imple-

mented on its basal formulation were capable of overcoming

the negative effect of lower winter temperatures and reduced

photoperiod in resin yield.

In spite of differences in site (C versus B) and year of

sampling (2004e2005 versus 2002e2003), consistent positive or

neutral effect was observed in resin yield of trees treated with

the individual metal adjuvants K, Fe and Cu in the presence of

CEPA. The positive effects on resin yield of CEPA and metal

supplemented paste in the two sites examined (B and C) indi-

cate a synergistic effect between ethylene and the metal

cofactors/activators of terpene synthases and ethylene

receptor. This was particularly evident when metals were

supplied individually. Combining two or more metals with

CEPA, however, often caused reduction in resin yield compared

to the basal paste control. This effect could be a consequence of

inefficient movement and/or access of combinedmetal ions to

transporters and sites of action in target enzymes and/or

b i om a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 4 4 4 2e4 4 4 8 4447

receptors in the presence ofCEPA. Competition betweenmetals

with similar properties for transporters and binding sites could

also have contributed to the decrease in resin yield in treat-

ments combining different metals.

The experiments carried out with individual metal adju-

vants devoid of CEPA in the paste showed that resin produc-

tion was equivalent to or better than that with CEPA

containing paste in site C. Since CEPA is the most expensive

component of the currently used commercial paste [19,21], its

complete or partial replacement by much cheaper metal

cofactors with identical or better resin yield from pine trees is

of great interest to the forest industry. One gram of CEPA is

over three thousand times more expensive than a gram of

potassium sulfate [37].

Although Pinus is regarded as a genus primarily dependent

on constitutively stored resin (within the complex resin duct

network) [20], the induced component of oleoresin formation

appears to be considerably important in tapped forests of P.

elliottii in southern Brazil. Significant responses to local

application of metal cofactors of terpene synthases at the first

season of tapping and yield stability over a year probably

reflect the relevance of the inducible component, which

would be consistent with the longevity of resin ducts and

secretory epidermal cells lining their lumen. In fact, detailed

studies on spruce species (another group of conifers regarded

as primarily dependent on constitutive resin-based defenses)

indicate that the inducible resin is very important for

responding to insect attacks and is correlated with the

induction of terpene synthases [38]; these data also put

methyl jasmonate (MJA) and MJA-induced ethylene at center

stage of resin-based defenses [28].

Trees treated with the metal supplemented pastes had

improved resin yield or, when without CEPA, an yield equiv-

alent to that of trees exposed to control-paste; however, resin

composition was not significantly changed. Most important,

the proportion of monoterpenes that act as main semi-

ochemicals for the interaction with bark beetles (a-pinene, b-

pinene andmyrcene) was not significantly changed in relation

to the control (Table 5).

The higher content of b-pinene in the oleoresin is favorable

to commercialization and is not usually found in P. elliottii;

previous reports on oleoresin composition of the species in

southeast Brazil found approximately 30% of b-pinene and

60% a-pinene (volume fraction of total turpentine terpenes)

[36]. However, even within the same species, monoterpene

composition may vary considerably, including the proportion

of a and b-pinene [29]. Another aspect to be considered that

may help explain this difference in a and b-pinene content is

the fact that the samples in this study were directly taken

from the freshly cut resin ducts and immediately frozen. The

maintenance of resin samples for several days in the field, as it

is usually done in studies of pine oleoresin composition (e.g

[36].), may cause profile alterations due to evaporation, air

oxidation and biotransformation by microorganisms, partic-

ularly in warmer climates. A survey of monoterpene compo-

sition (both emission and tissue internal concentration)

conducted with major forest species in the USA showed that

a and b-pinene were the most abundant monoterpenes in

Pinus species [39]. P. elliottii available data for the USA indicates

an average (% in total monoterpenes) of 47.3 a-pinene and 37.7

b-pinene. The same species showed a proportion of 52 b-

pinene and 22 a-pinene in leaf extracts, whereas for leaf

emissions these values were 37 and 48, respectively. Some

pine species showed higher b-pinene % of monoterpenes

compared to a-pinene, such as Pinus clausa, Pinus palustris and

Pinus radiata [39], Pinus contorta [25] e Pinus ponderosa [40].

The absolute concentration of both pineneswas reduced to

approximately a third of the control levels in the resin of trees

exposed to 1 mol m�3 Mn. No major change in the profile of

other terpenes was observed, suggesting that Mn is probably

activating other enzymes and driving the flux of carbon to

other metabolites. The increase in camphene content in the

oleoresin of metal adjuvant exposed-trees may indicate that

these cofactors lead to a higher activation of terpene syn-

thases catalyzing the synthesis of the bornyl and camphyl

cation compared to those enzymes leading to the pinyl cation

[41]. Alternatively, the pinene synthases may limit availability

of metal cofactors to enzymes involved in the assembly of

minor components of the oleoresin. The trend toward a slight

reduction in pinene concentration in the oleoresin may also

reflect the activation of the other synthases that use precur-

sors common to pinene synthases.

4. Conclusion

Metal cofactors of terpene synthases and of ethylene recep-

tors, applied locally as stimulant paste on wounded bark

tissue, improve slash pine oleoresin yields without causing

significant changes in its composition. Some adjuvants are

capable of replacing ethylene-releasing products, currently

used in commercial stimulant pastes, supporting equivalent

oleoresin yields. This feature can significantly reduce the

costs of resin tapping activity in P. elliottii. The capacity to

sustainably modulate oleoresin yield with metal adjuvants

starting from the first year of tapping indicates that the

induced component of oleoresin in Pinus trees is likely more

important than previously thought.

Acknowledgments

Financial support from Ambar Florestal Ltda. e Sao Jose do

Norte e RS - Brazil (grant in aid of research) and the Brazilian

Research Funding Agencies CNPq and CAPES is gratefully

acknowledged. The authors thank Ambar staff for support in

the field work.

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