Planta (2023) 257:19

https://doi.org/10.1007/s00425-022-04050-7

REVIEW

Laticifer ontogenesis and the chemical constituents of Marsdenia

zehntneri (Apocynaceae) latex in a semiarid environment
Hellen Karla Oliveira Marques1 · Maria Gabriela Ferreira Figueiredo1 · Willian Samuel de Souza Pio1 ·
Leonardo Monteiro Ribeiro1 · Islaine Franciely Pinheiro de Azevedo1 · Lucienir Pains Duarte2 ·
Grasiely Faria de Sousa2 · Mariana Guerra de Aguilar2 · Maria Olívia Mercadante‑Simões1 

Received: 9 November 2022 / Accepted: 8 December 2022 / Published online: 20 December 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Main conclusion  Anastomosed laticifers with intrusive growth produce latex containing methyl comate and betulin
with economic and ecological value in arid environments. Climatic factors influence laticifer development in the api-
cal meristem and vascular cambium.

Abstract  Latex is a complex emulsion with high medicinal as well as ecological value related to plant survival. Marsdenia
zehntneri is a shrubby plant that grows on limestone outcrops in the semiarid regions of Brazil. We sought to characterize the
ontogenesis of the laticifers of this species and to relate that process to climatic seasonality and phenology through anatomi-
cal, ultrastructural, and micro-morphometric evaluations of the apical meristem and vascular cambium. The histochemistry
of the secretory structure was investigated and the chemical composition of the latex was analyzed. Phenological assessments
were performed by monitoring phenological events for 1 year. The laticifers network of M. zehntneri permeates the entire
primary and secondary body of the plant, providing a wide distribution system of defensive compounds. Its laticifers, of a
distinct mixed type (anastomosed, with intrusive growth), are numerous and voluminous in the apical meristem but scarce
and minute in the secondary phloem. Latex secretion involves the participation of oleoplasts, polysomes, and dictyosomes.
Methyl 2,3-dihydroxy-ursan-23-oate, methyl 3-hydroxy-ursan-23-oate, and betulin are encountered in high proportions in
the latex and have ecological and medicinal functions. The development of primary laticifers is related to the resumption of
apical meristem activity with increasing day length at the end of the austral winter. The development of secondary laticifers
is related to high summer temperatures and rainfall that favor vascular cambium activity. The wide distribution of laticifers,
their seasonal pattern of secretion, and their latex composition contribute to the adaptation of M. zehntneri to its natural
environment.

Keywords  Apical meristem · Climatic seasonality · Latex constituents · Oleoplasts · Phenology · Secondary phloem

Introduction

Laticifers compose a complex secretion system permeating

Communicated by Anastasios Melis.
* Maria Olívia Mercadante‑Simões
omercadante@hotmail.com
1
General Biology Department, Centro de Ciências Biológicas
e da Saúde, Universidade Estadual de Montes Claros,
Montes Claros, Minas Gerais CEP 39401‑089, Brazil
2
Chemistry Department, Instituto de Ciências Exatas,
Universidade Federal de Minas Gerais, Belo Horizonte,
Minas Gerais CEP 31270‑901, Brazil
latex-producing plants (Fahn 1979; Mahlberg 1993). Latex
exudation is observed throughout cross-sections of the stems
in the primary structures of Apocynaceae species, as well
as in the inner region of the bark in secondary structures
(Gonçalves et al. 2018; Souza et al. 2021). Laticiferous
ducts take on several structural forms that can be correctly
identified only through ontogenetic studies of their precur-
sor cells, with profound changes occurring early and over
only short periods of time (Gama et al. 2017; Teixeira et al.
2020; Gonçalves et al. 2018; Almeida et al. 2021; Souza

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et al. 2021). Most of the laticifers described in Apocynaceae
species are of the anastomosed type, in which adjacent cell
walls dissolve to form a branched system of latex-secreting
ducts (Lopes et al. 2009; Gama et al. 2017; Gonçalves et al.
2018; Souza et al. 2021); intrusive growth processes have
also been reported, however, where cell ends elongate in
the midst other surrounding meristematic cells (Canaveze
and Machado 2016; Canaveze et al. 2018). Considering the
wide diversity and geographic distribution of the Apocyn-
aceae family, detailed studies of their laticifers will contrib-
ute to the expansion of knowledge concerning the complex
ontogenesis of those secretory structures.
Latex is an emulsion (a natural colloidal solution) rich
in various hydrophilic and lipophilic chemical compounds
produced by both primary and secondary metabolism (Lopes
et al. 2009; Ramos et al. 2019). The secretion of latex com-
ponents occurs early in the precursor cells of laticifers, and
normally involves the presence of oleoplasts, endoplasmic
reticulum, dictyosomes, and cytosol, associated with the
presence of a bulky nucleus, mitochondria, and amyloplasts
involved in the energetically demanding processes of latex
synthesis (Gonçalves et al. 2018; Souza et al. 2021). The
chemical compounds present in latex play decisive roles in
a plant's adaptation to its environment, offering resistance to
herbivory and pathogens as well as wound sealing by coagu-
lation when in contact with air (Farrel et al. 1991; Hart-
mann 2004; Agrawal and Konno 2009; Konno 2011; Freitas
et al. 2016; Ramos et al. 2019). Nonetheless, and despite the
importance of this topic, there is little information avail-
able associating latex production with environmental factors
under semiarid environmental conditions.
The activities of the apical and lateral meristems, includ-
ing the formation of laticifers, latex production in meris-
tematic regions, and vegetative as well as reproductive
phenological events, are commonly conditioned by climatic
variations during the passage of the seasons (Marcati et al.
2016; Tarelkin et al. 2019; Angyalossy et al. 2020). Spe-
cies of the genus Marsdenia R. Br occupy a great diversity
of environments, and a large percentage of those species
grow in dry regions and have restricted distributions. Mars-
denia zehntneri is a shrubby species endemic to the semiarid
regions of Brazil. Its roots penetrate shallow but fertile soils
in deep cracks in limestone outcrops in environments with
high temperatures, strong incident sunlight, and water avail-
ability concentrated into short episodes (Rapini and Pereira
2011; BFG 2022; Espírito Santo et al. 2018). Masrsdenia
species are latex producers, and many of them have medici-
nal value (Sheng-Xiang et al. 1993; Shikwambi et al. 2021;
Chen et al. 2022; Lin et al. 2022; Wu et al. 2022), although
no in-depth information on the synthesis and composition of
M. zehntneri latex is available in the literature.
We therefore examined the ontogenetic processes of the
laticifers in the apex and in vascular cambium of the stems of
13
Planta (2023) 257:19
M. zehntneri and performed chemical analyses of the latex.
Additionally, considering the ecological value of its latex
and the occurrence of this species in rocky environments
in semiarid regions, we tested the hypothesis that latex pro-
duction has adaptive value for those conditions by posing
the following questions: (1) How are laticifers distributed in
the plant body? (2) What is their structural type? (3) How
do the ultrastructural aspects of the laticifers relate to their
structural types and latex secretory processes? (4) What are
the relationships among the main chemical constituents of
latex and their biological activities and ecological functions?
(5) What are the effects of climatic seasonality on laticifer
formation and phenology?
Materials and methods
Plant material
Adult individuals of M. zehntneri are erect shrubs, 1–2.5 m
tall, that grow on shallow soils and in deep crevices on lime-
stone outcrops (Fig. 1a). When damaged, the vegetative apex
exudes a milky white latex, especially in the leaf abscission
region (Fig. 1b). The leaves are lanceolate and glabrous, and
the inflorescence is umbelliform, with flowers with yellow
corollas (Fig. 1c). The fruits are elongated, fusiform, and
glabrous mericarps, and the seeds have plumose filiform
appendages (Fig. 1d).
The plant material for examination consisted of newly
developed shoot apices, and was collected monthly between
March/2019 and February/2020 from five individuals of M.
zehntneri occurring in a natural population in the Brazilian
Cerrado (neotropical savanna) (16°45′42.7′′ S 43°54′41.9′′
W). That region has a semiarid climate, with strong seasonal
rainfall that is concentrated in the austral summer (Novem-
ber–February). Reference specimens with fertile branches
were deposited in the Norte Mineiro Herbarium—MCCA, of
the Institute of Agrarian Sciences, of the Federal University
of Minas Gerais, Brazil (MCCA 2557).
Structural analyses
Samples from the basal portion of the shoot apex, contain-
ing bark and the peripheral region of the xylem, were fixed
in Karnovsky's solution (Karnovisky 1965), under vacuum
(560 mm Hg) for 24 h, dehydrated in an ethanol series, and
subsequently embedded in hydroxymethyl-methacrylate
resin (Leica Microsystems Inc., Heidelberg, Germany).
Cross and longitudinal sections (5 μm thick) were prepared
using a rotary microtome (Atako, Tokyo, Japan), stained
with toluidine blue pH 4.7, and mounted on permanent
slides with acrylic resin (Itacril, Itaquaquecetuba, Brazil).
Planta (2023) 257:19 Page 3 of 20  19
Fig. 1  General aspects of Marsdenia zehntneri Fontella (Apocyn-
aceae). a Adult individuals (arrows) on a limestone outcrop (arrow-
head). b Vegetative apex (arrow), latex in the region of leaf abscis-
Micromorphometry
Cross-section samples were evaluated to measure vascular
cambium thickness (defined as the region between the newly
differentiated secondary phloem cells and vessel elements).
Ten measurements were taken at equidistant points, total-
ing 50 measurements of each of the 5 individuals evalu-
ated, using Axion Vision LE Rel.4.8.2 software version 15.0
06–2010 (Zeiss, Jena, Germany). Data were submitted to
analysis of variance and the means compared using Tukey's
test (P < 0.05%).
Ultrastructural analyses
Samples from the distal region of the shoot apex were
fixed in Karnovsky's solution (Karnovisky 1965) for 24 h,
post-fixed with 1% osmium tetroxide (0.1 M phosphate
buffer, pH 7.2), dehydrated in an acetone series, and sub-
sequently embedded in Araldite resin (Leica Microsys-
tems, Heidelberg, Germany). Ultrathin sections (50 nm
thick) were obtained using a UC6 ultramicrotome (Leica
Microsystems, Heidelberg, Germany), contrasted in
sion (arrowhead), and lanceolate leaves. c Umbel and flower (arrow).
d Fruit mericarp, elongated, fusiform, and glabrous (arrowhead) and
seeds with feathery filiform appendages (arrowhead)
a saturated solution of uranyl acetate and lead citrate
(Roland 1978), and examined in a CM 100 transmission
electron microscope (Philips/FEI Corporation, Eindhoven,
The Netherlands) at 80 kV.
Histochemistry
Samples obtained as described above (structural evalua-
tions) were subjected to the following histochemical tests:
NADI (α-naphthol and dimethylparaphenylene diamine
hydrochloride) (David and Carde 1964) for terpenoids;
Lugol's reagent (Johansen 1940) for alkaloids; Toluidine
blue pH 4,7 (O'Brien et al. 1964 modified) for phenolic
compounds; DMACA (p-dimethylaminocinnamaldehyde)
(Feucht et al. 1986) for flavonoids; XP (Xylidine-Poinceau)
(Vidal 1970) for proteins; and ruthenium red (Johansen
1940) for acidic polysaccharides. Control tests were per-
formed simultaneously according to the recommendations
of the respective authors. Photographic documentation was
performed using an AxionCam ICC.
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Page 4 of 20
Chemical analysis
Extracts and phytochemical prospection Samples of M.
zehntneri latex were collected from five individuals in
October, which is a rainy and warm period in the Brazil-
ian Cerrado region. These samples were pooled (642.2 mg)
and subjected to extraction with hexane, chloroform, ethyl
acetate, methanol, and water (four 10 mL portions of each
extracting solvent), previously distilled. After filtration and
complete removal of the solvent, hexane (LHE 380.2 mg),
chloroform (LCE, 56.8 mg), ethyl acetate (LEAE, 5.9 mg),
methanolic (LME, 95.8 mg), and aqueous (LAE, 66.0 mg)
extracts were obtained. The dry LAE was obtained after
freeze-drying. At the end of all of the extraction processes, a
residue (R, 32.4 mg) insoluble in all extracting solvents was
obtained (Fig. S1 Supplementary material). The dry extracts
and the R residue were analyzed using different techniques
to identify classes of metabolites and their main constituents.
Thin-layer chromatography The classes of metabolites
present in the extracts of M. zehntneri latex were identified
by thin-layer chromatography (TLC) using specific stain-
ing reagents according to Wagner and Bladt (1996) proce-
dures. Chromatoplates were prepared using a suspension of
silica gel 60G in water, deposited on glass plates forming a
0.25 mm-thick layer. After partial drying at room tempera-
ture, the chromatoplates were activated at 100 ºC for at least
30 min. For TLC analysis, each extract was dissolved in
the same solvent used in the extraction process. The detec-
tion of terpenoids, phenylpropanoids, lignans, and saponins
was carried out by the appearance of spots in the visible
with diverse coloration (purple, pink, blue, or yellow) after
spraying the plate with a 1:1 v/v solution of vanillin (in ethyl
alcohol; 1:99) and perchloric acid (in water; 3:97), followed
by heating at 100 ºC. The presence of lignans was also inves-
tigated by UV fluorescence. Flavonoids were detected by
autofluorescence in the ultraviolet (365 nm) after spraying
2% diphenylboryloxyethylamine (NP) in methanol (w/v), fol-
lowed by spraying 5% polyethylene glycol in ethanol (PEG-
4000) (w/v). The presence of alkaloids was confirmed by
brown staining observed after treatment of plates with Dra-
gendorff's reagent (1 mL of 1:1 solution of 0.85 g bismuth
nitrate in 10 mL glacial acetic acid and 8 g potassium iodide
in 30 mL water added to 2 mL glacial acetic acid and 10 mL
water), followed heating at 100 ºC. Amino acids, biogenic
amines, and ephedrines were detected by orange-colored
spots observed after spraying ninhydrin solution (30 mg of
ninhydrin dissolved in 10 mL of n-butanol and 0.3 mL of
98% acetic acid) and heating at 100 ºC. The presence of
anthraquinone was confirmed by red spots under visible light
and yellow spots under UV light (365 nm). And the detec-
tion of coumarins was confirmed by blue spots generated
after treatment with 5% potassium hydroxide in ethanol. The
eluents (v/v) and staining reagents used to identify classes
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Planta (2023) 257:19
of metabolites of M. zehntneri latex by TLC are presented
in Table S1 (Supplementary material).
Infrared spectroscopy, nuclear magnetic resonance,
and gas chromatography coupled to mass
spectrometry
The infrared (IR) spectra were recorded on a ­Shimadzu®
IR-408 spectrometer with samples in KBr [1% (m/m)] pel-
lets. The 1H and 13C-NMR spectra recorded at 400.129 and
100.613 MHz, respectively, were performed on a Bruker®
model Avance DRX-400. The deuterated solvents used are
indicated in their respective NMR spectra. The chemical
shifts (δ) were registered in ppm using tetramethylsilane
(TMS) as internal reference standard. The coupling con-
stants (J) were registered in Hertz (Hz). Gas chromatogra-
phy coupled to mass spectrometry (GC–MS) analyses were
performed on a GCMS-QP2010 ULTRA chromatograph
­(Shimadzu®). The analysis conditions were: 2 µL injection
volume; injector at 320 °C Split (1: 50), column Rxi-1MS
(30 m × 0.25 mm × 0.25 µm) Restek®; column temperature
60 °C (5 min), followed by 10 °C/min, until 300 °C; inter-
face GC–MS at 320 °C; MS detector (Electronic impact at
70 eV) at 320 °C; Helium at 3.0 mL/min as carrier gas. The
compounds were putatively identified by comparative analy-
sis of fragmentation data from the mass spectra using soft-
ware (GC–MS Postrun Analisys, v.4.20, LabSolutions, Shi-
madzu) by comparison with the Spectral Library NIST147,
NIST27 (National Institute of Standards and Technology,
Department of Commerce, USA) and ­Wiley® 7.
Climatic seasonality and phenology
The study region has strongly seasonal rainfall concentrated
in the austral summer, between November and February (an
Aw climate, according to the Köppen classification system;
Antunes, 1994) (INMET 2022). Day length, average tem-
peratures, and monthly rainfall data were obtained from the
website of the National Institute of Meteorology—INMET/
BR (https://​portal.​inmet.​gov.​br).
Ten individuals at reproductive maturity with current
and previous signs of reproduction were selected for phe-
nological evaluations. Those individuals were evaluated
monthly, from March/2019 to February/2020, and their
vegetative (production of young, mature, and old leaves)
and reproductive (floral buds, open flowers, immature,
ripe, and dispersing fruits) phenophases estimated in
percentages. Circular statistics were performed using the
Oriana program to assess the seasonality of their vegeta-
tive and reproductive phenophase patterns (Kovach 1994),
calculating their circular standard deviation, circular mean
(μ), and the length of the mean vector (r) that represents
how the data are clustered around the mean (0—uniformly
Planta (2023) 257:19 Page 5 of 20  19
distributed, 1—clustered). The Rayleigh test was then per-
formed, where p < 0.05 and r > 0.5 indicate a unimodal
distribution (and therefore seasonality) in an observed
phenophase. The months were converted into angles at
intervals of 30º, with 0º = January, successively up to
330º = December. To determine the influence of climatic
variables (day length, temperature, and precipitation—pre-
dictor variables) on the evaluated phenophases, multiple
regressions in the vegan package (Oksanen et al. 2013)
were run in the R program. Previously, the relationships
between variables were tested using Spearman correla-
tions. As the variables showed low correlation (Spearman
correlations: rs = 0.37–0.68; n = 12 months), all of the
environmental variables mentioned above were included in
the model. Akaike Information Criterion (AIC) was used
Fig. 2  Anastomosed laticifers on the shoot apex of Marsdenia zehnt-
neri Fontella (Apocynaceae) evidencing intrusive growth. Longitu-
dinal sections. a, b Stem apex with laticiferous system (arrowhead)
differentiated early in the regions of the ground meristem and pro-
cambium, concentrated in the region of the knodes, and present in the
interior of colleters. b Box detail in a. c-d Laticifers of the anasto-
to select the simplest and most parsimonious model sup-
ported by the data (Burnham and Anderson 2002).
Results
Distribution and structural types of laticifers
in primary and secondary structures
The shoot apex has a laticiferous system with early differen-
tiation in ground meristem and procambium regions. Latic-
ifers are concentrated in the region of the knodes and also
present within colleters (Fig. 2a, b). The laticifers evidenced
a mixed anastomosed structural type, with intrusive growth;
they are elongated, with acuminate ends, and have lateral
projections. Laticifer walls are rich in pectic compounds,
mosed mixed structural type with intrusive growth, elongated, with
acuminate ends and lateral projections (arrow). Walls rich in pectic
compounds (stained pink with toluidine blue pH 4.7), dense cyto-
plasm, voluminous nuclei, elongated within the intercellular spaces.
co colleter, lp leaf primordium, pc procambium, pi pith, pm promeris-
tem, sk stem knode
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Page 6 of 20
have a dense cytoplasm, a voluminous nucleus, and elon-
gate within intercellular spaces rich in pectic compounds
(Fig. 2c, d). The primary structure has laticifers in the cortex
between the phloem strands of the bicolateral bundles and
in the pith (Fig. 3a–d). Laticifers have sinuous walls due
to their intrusive growth between and among parenchyma
cells (Fig. 3e, f). In the secondary structure, the laticifers
are associated with companion cells and sieve tube elements
(4a–c), and are non-articulated, minute, quite short, and of
small calibers (4d–i).
Ultrastructure and secretory processes
Laticifers show intrusive growth at their ends, with loose cell
walls having pectic natures (Fig. 5a). During the secretory
process, it is possible to identify cellular components related
to the synthesis of the compounds identified in the histo-
chemical tests: plastids related to terpene droplet synthesis,
dictyosomes related to acidic polysaccharide synthesis, and
rough endoplasmic reticulum and polysomes related to pro-
tein synthesis (Fig. 5a–c). Additionally, starch grains and
numerous mitochondria related to the high-energy demands
of latex synthesis processes can be identified. Laticifers have
peripheral cytoplasm and a large central vacuole associated
with laticifer maturity and latex accumulation (Fig. 5b–d).
Chemical compounds
Histochemistry and TLC phytochemical profile Histochemi-
cal tests of the laticifers revealed the presence of terpenoids
stained lilac with NADI (Fig. 6a, b), alkaloids stained brown
by Lugol (Fig.  6c), flavonoids stained red by DMACA
(Fig. 6d), protein stained red by XP (Fig. 6e), and pectic
compounds stained pink by ruthenium red (Fig. 6f).
The following classes of organic compounds were
detected in the M. zehntneri extracts using TLC analyses:
terpenoids and phenylpropanoids in LHE, LCE, LEAE,
and LME; saponins, alkaloids, and flavonoids in LME; and
amino acids, biogenic amines, and/or ephedrines in LAE
(Table S2). Lignans and coumarins were not detected by
TLC in latex extracts of M. zehntneri.
IR spectroscopy Through the infrared spectroscopic anal-
yses, several functional groups, associated with a chemical
diversity in the extracts and in the residue obtained at the
end of the extractions of M. zehntneri latex, were observed.
In the IR spectra, characteristic bands (Figs. S2–S7) of the
classes of metabolites detected by TLC (Table S2) were
observed.
All the IR spectra obtained show bands between 3386
and 3442 ­cm−1, characteristic of the stretching of hydroxyl
groups (O–H). In the spectra of LHE (Fig. S2), LCE (Fig.
S3), LEAE (Fig. S4), LME (Fig. S5), LAE (Fig. S6), and
of the residue R (Fig. S7), absorption bands between 2926
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Planta (2023) 257:19
and 2854 ­cm−1 were observed, referring to the C–H-bond
stretching of methyl groups ­(CH3) and methylene groups
­(CH2). Bands between 1718 and 1734 ­cm−1, characteristic of
the C=O stretching of carbonyl groups, were observed in the
spectra of LHE, LCE and LEAE. Moreover, in the spectra of
these three extracts, were also observed bands at 1376 ­cm−1
and at 1458 ­cm−1 characteristic of angular deformation of
the C–H bond of methyl and methylene groups (Barbosa
2007). The IR spectrum of LME (Fig. S5) showed bands at
1660  ­cm−1 and at 1394 ­cm−1 characteristic of C=C-bond
stretching and O–H-bond angular deformation, respectively.
This spectrum also showed the band at 1076 ­cm−1 refer-
ring to C–O-bond stretching and bands between 668 and
956 ­cm−1 corresponding to out-of-plane angular deforma-
tion of C–H bonds of aromatic compounds (Barbosa 2007).
The IR spectrum of the LEAE extract (Fig. S6) shows bands
between 2926 and 2856 ­cm−1, referring to the C–H stretch-
ing of methyl groups (­ CH3) and methylene groups (­ CH2) and
a band at 1734 ­cm−1 characteristic of the C=O stretching of
carbonyl groups.
The IR spectrum of the final residue (Fig. S7) obtained
after all the extractions of the latex of M. zehntneri showed
a band at 3040  ­cm−1, referring to the stretching of the
H–C=C bond of alkenes, as well as bands at 2924 ­cm−1 and
2854 ­cm−1 referring to the stretching of the C–H bonds of
methyl groups (­ CH3) and methylene groups (­ CH2). A band
at 1654 ­cm−1 characteristic of C=C-bond stretching, bands
at 1450 ­cm−1 and 1376 ­cm−1 typical of C–H-bond angular
deformation of methyl and methylene groups and a band at
836  ­cm−1 correspondent to H–C=C-bond stretching were
also observed. These absorption bands are characteristic of
polyisoprene formed from the cis-1,4-polyisoprene isomer
(Barbosa 2007; Dghim et al. 2015).
NMR data of extracts
The profiles of the 1H NMR, the 13C-NMR spectra of the
LHE, LCE and LEAE, LME and LAE are presented in the
Supplementary material (Figs. S8–S16).The profiles of the
1
H NMR spectra of the LHE, LCE (Figs. S8, S9), and LEAE
(S11) were very similar (Figs. S8–S16). For example, in
the spectrum of LCE, there are two singlets at δH 1.6 and
δH 2.0, characteristic of allylic hydrogen, and the signal at
δH 5.1 which is characteristic of olefinic hydrogen. In the
13
C-NMR spectrum of LCE (Fig. S10), the signals at δC
23.5, δC 26.5 and δC 32.3 are characteristics of aliphatic
carbons and the signals at δC 135.3 and δC 125.2 are typical
of olefinic carbons. By comparing this set of signals with
those reported by Dghim et al. (2015), it was possible to
confirm the presence of the cis-1,4-polyisoprene isomer. In
general, polyisoprene is detected in apolar extracts and is
the precursor of polymers found in latex produced by plants
(Kalita and Saikia 2004). In the 1H NMR spectra of the
Planta (2023) 257:19 Page 7 of 20  19

Fig. 3  Articulated laticifer with intrusive growth in the primary stem
structure of Marsdenia zehntneri Fontella (Apocynaceae). a, b Cross-
sections. c–f Longitudinal sections. a–d Laticifers (arrows) distrib-
uted in the cortex, between the phloem strands of the bicolateral bun-
dles and in the pith (phenolic compounds of latex stained blue with
toluidine blue pH 4.7). e–f Laticifers with sinuous walls (arrowheads)
because of parenchymal cell detachment during intrusive growth
(latex alkaloids stained brown by Lugol). co cortex, dr druse, gf
gelatinous fibers, ip inner phloem, op outer phloem, ph pheloderm, pi
pith, xy secondary xylem

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Page 8 of 20
Fig. 4  Laticifers in the secondary stem structure of Marsdenia zehnt-
neri Fontella (Apocynaceae). Cross-sections. a panoramic view of
the stem. b, c Laticifers (arrowheads) associated with companion
cells and phloem sieve tube elements. d–i Laticifers (arrowheads)
not articulated, minute, with small calibers and lengths. a–c Acidic
polysaccharides in the latex stained pink by toluidine blue pH 4.7.
LHE, LCE (Figs. S8-S9), and LEAE (S11), it was also pos-
sible to observe signals between δH 0.7 to δH 1.7 correspond-
ing to hydrogen atoms of aliphatic hydrogen chain -CH2 and
-CH3 groups. The signals between δH 4.4 and δH 5.5 are
attributed to olefinic hydrogen atoms, which are commonly
found in latex samples. These observed signals are typical
of terpenoid compounds. In the 1H NMR spectrum of LME
(Figs. S12, S13), the signals between δH 0.7 and δH 1.7 are
characteristics of aliphatic hydrogens. The signals between
δH 2.8 and 4.0 are attributed to hydrogen atoms attached
to oxygenated carbon and the signals between δH 5.0 and
5.7 are attributed to olefinic and anomeric hydrogen atoms
of sugars. In addition, signals were observed in the region
of δH 7.0–7.9 that are characteristic of aromatic hydrogen
atoms. These signals are typical of phenolic compounds
and mucilages. In the 1H NMR spectrum of LAE (Figs.
13
Planta (2023) 257:19
d–i Latex alkaloids stained brown, and starch grains stained lilac by
Lugol. ca cambium, cc companion cell, co cortex, gf gelatinous fib-
ers of primary phloem, ip inner primary phloem, ph phelloderm, pi
pith, ra ray, sp secondary phloem, si sieve tube element, xy secondary
xylem
S14-S15), signals were observed in the regions of aromatic
(δH 6.6–8.0), region of hydrogen atoms’ characteristics of
amino acids (δH 2.5–3.9) and also in the region of hydrogen
atoms of carbinolic (δH 4.0–4.6) hydrogen atoms. These sig-
nals are typical of proteins, alkaloids, and mucilages. The
signals detected by NMR corroborate the phytochemical
profile of M. zehntneri extracts obtained by TLC (Table S2).
GC–MS analysis of extracts The volatilizable metabo-
lites of M. zehntneri latex and their relative amounts were
identified by GC–MS of LHE, LCE, and LEAE (Fig. S17
and Table 1). The main constituents identified correspond
to three pentacyclic triterpenes being two of them of ursane
skeleton [methyl 2,3-dihydroxy-ursan-23-oate, methyl
3-hydroxy-ursan-23-oate] and one of lupene skeleton [betu-
lin] (Fig. 7). These compounds account for more than 57%
of the substances identified. A diterpene, ethers, long-chain
Planta (2023) 257:19 Page 9 of 20  19
Fig. 5  Ultrastructural aspects of the intrusive growth of laticifers
and latex synthesis in the apical stem meristem of Marsdenia zehnt-
neri Fontella (Apocynaceae). a, b Intrusive growth, loose cell walls
(arrowheads). a–c Plastids, dictyosomes, rough endoplasmic reticu-
lum and polysomes related to terpene, acidic polysaccharides, and
protein synthesis. b–d Numerous starch grains and mitochondria
esters, a long-chain alcohol, hydrocarbons, and fatty acids
were also identified (Fig. 7 and Table 1).
Climatic seasonality, annual cambium activity,
and phenology
The shortest day lengths during the study period (from
March/2019 to February/2020) occurred in June and July
(Fig. 8a). The rainy season began in September, and the
month with the highest rainfall rate was January (Fig. 8b).
The highest temperatures were recorded in October and
November, and the lowest in June and July (Fig. 8b).
Climatic components affect the vascular cambium, api-
cal meristem, and phenophases differently. The vascular
cambium, which produces phloem and secondary latic-
ifers, is active when temperatures and precipitation are
related to the high-energy demands of latex synthesis processes. d
Peripheral cytoplasm and a central and bulky vacuole related to latex
accumulation (arrowhead, intrusive growth). cl chloroplast, cv cen-
tral vacuole, cw cell wall, di dictyosomes, er endoplasmic reticulum,
mi mitochondria, nu nucleus, pc peripheral cytoplasm, pl plastid, st
starch grain, te terpenoids
decreasing (Fig. 8b, c). Vascular cambium activity begins
with the production of xylem between March and April
(Fig. 9a); phloem, where the laticifers develop, differenti-
ates later, between May and June (Fig. 9b). The vascular
cambium remains inactive during the late dry season and
the entire rainy season (Fig. 9c, d).
Apical meristem activity is triggered by increasing day
length, with leaves, flowers, and primary laticifers being
produced during the rainy season (Fig. 10). The occur-
rences of new leaves and mature leaves were seasonal (new
leaves: Z = 100.79, p < 0.01, r = 0.60) with mean angles in
November and March, respectively. New leaf production
correlated significantly and positively with temperature
(F = 6.24; R2 = 0.32; p < 0.03) (Fig. S17).
Regarding the reproductive phenophases, both bud and
flower production showed marked seasonality, with average
13
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Page 10 of 20 Planta (2023) 257:19

Fig. 6  Chemical compounds in the stem laticifers of Marsdenia zehntneri Fontella (Apocynaceae). a, b Terpenoids (purple, NADI), c alkaloids
(brown, lugol), d flavonoids (red, DMACa), e proteins (red, XP), and f pectic compounds in cell walls and cell contents (pink, ruthenium)

dates in October and November, respectively, during the Green (Z = 33.31, p < 0.01, r = 0.68), ripe (Z = 35.51,
rainy season (buds: Z = 47.73, p < 0.01, r = 0.97; flowers: p < 0.01, r = 0.80), and dispersed (Z = 10.19, p < 0.01,
Z = 3, p < 0.05, r = 1). Flower bud production was positively r = 0.59) fruits occurred in the dry season, with average dates
associated with temperature (F = 4.58; R2 = 0.25; p < 0.05). in April, July, and August, respectively. The occurrence of

13
Planta (2023) 257:19 Page 11 of 20  19

Table 1  Compounds identified Peak % Area Compound Molecular formula Similarity (%)

by GC–MS in hexanic (LHE),
chloroform (LCE), and ethyl LHE
acetate (LEAE) extracts of
 1 0.83 Caur-16-ene C20H32 86
Marsdenia zehntneri latex
 2 0.58 1,2-epoxy-hexadecane C16H32O 86
 3 1.58 1,2-epoxy-octane C18H36O 87
 4 1.15 (Z,Z)-3,13-octadecadien-1-ol C18H34O 76
 5 55.73 Methyl 2,3-dihydroxy-ursan-23-oate C31H50O4 86
 6 35.04 Methyl 3-hydroxy-ursan-23-oate C31H50O3 89
 7 5.09 Betulin C30H50O2 86
LCE
 1 9.39 Octyl 10-undecenoate C19H36O2 78
 4 4.62 Pentatriacontane C35H72 94
 5 7.30 Tetracontane C40H82 95
 14 31.70 Methyl 2,3-dihydroxy-ursan-23-oate C31H50O4 87
 15 21.41 Methyl 3-hydroxy-ursan-23-oate C31H50O3 89
 18 4.04 Betulin C30H50O2 83
LEAE
 1 7.92 Oleic acid C18H34O2 84
 2 43.43 Methyl 2,3-dihydroxy-ursan-23-oate C31H50O4 84
 3 48.65 Betulin C30H50O2 83

ripe fruits was negatively associated with the photoperiod
(F = 20.75; R2 = 0.64; p < 0.01) (Fig. S17).
Discussion
Marsdenia zehntneri is a latescent shrub that grows on lime-
stone outcrops under a semiarid climate where it is exposed
to extensive periods of drought. We characterize here the
distribution of its laticifers, their developmental pattern,
ultrastructural aspects of the secretory process, and the
chemical composition of its latex. An integrated evaluation,
considering phenological and climatic information, as well
as comparisons with other latex-producing species of Apo-
cynaceae that occur in the same environment, allow us to
discuss the adaptive value of latex production.
How are laticifers distributed?
We observed that the laticifers of M. zehntneri originate
early in the fundamental meristem and procambium, later
arising from fusiform cambial derivatives of the phloem.
Laticifers developing from stem promeristem cells (Gon-
çalves et al. 2018; Souza et al. 2021) occur throughout the
cortex, pith, and primary phloem in several species of the
family (Baratto et al. 2010; Gama et al. 2017; Canaveze
and Machado 2016). Laticifers can be observed between
pericyclic fibers, within the cortex, and in the medullary
rays amidst phloem strands (Salomé et al 2022). Colleters,
recorded here in M. zenhtineri, protect developing leaf
primordia in the apical meristem (Mercadante-Simões
and Paiva 2013). Those secretory structures are frequently
observed in Apocynaceae species, and may contain latic-
ifers within their central column (Thomas and Dave 1989;
Appezzatto-da-Glória and Estelita 2000).
In the secondary structure, the laticifers of M. zehntneri
originate from cambial fusiform phloem derivatives, as has
been reported in other species of the family (Nakazawa et al.
2013, Gonçalves et al. 2018; Souza et al. 2021; Salomé
et al. 2022). The association of laticifers with sieve tube
elements may favor the availability of carbohydrates that
are necessary to supply the high-energy demands of latex
synthesis. Contrary to what was observed in M. zehntneri,
laticifers can occur in phloem and xylem rays (Sando et al.
2009; Canaveze and Machado 2016), where they provide
additional protection to the internal tissues of the stem. The
laticifer network permeates the entire primary and second-
ary body of the plant, providing a broad delivery system of
defensive compounds.
What is the structural type of laticifers?
The laticifers of M. zehntneri are structurally anastomosed
and grow intrusively. For the correct identification of the
structural type of a laticifer, their precursor cells must be
examined among the meristematic cells at the elongating
apex or among the meristematic cells derived from the vas-
cular cambium (Gama et al. 2017; Gonçalves et al. 2018;
Souza et al. 2021; Teixeira et al. 2020), Salomé et al 2022).
Anastomosed articulated laticifers, as well as laticifers with

13
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Page 12 of 20 Planta (2023) 257:19

Fig. 7  Chemical compounds identified by GC–MS in the hexanic (LHE), chloroform (LCE), and ethyl acetate (LEAE) extracts of Marsdenia
zehntneri Fontella (Apocynaceae) latex (Apocynaceae)

intrusive growth originate, respectively, from the dissolu- structurally articulated anastomosed type (Lopes et al. 2009;
tion of adjacent cell walls or elongation through intercellu- Gama et al. 2017; Gonçalves et al. 2018; Souza et al. 2021;
lar spaces (Fahn 1979; Malberg 1993; Johnson et al. 2021). Salomé et al. 2022). Those laticifers guarantee a voluminous
Most laticifers among Apocynaceae species are of the exudation of latex that will flow to the region of mechanical

13
Planta (2023) 257:19 Page 13 of 20  19
Fig. 8  Climatic seasonality in Marsdenia zehntneri Fontella (Apo-
cynaceae). a Increasing day length from July. b Rainy season and
high temperatures from November to March. Dry season from April
or biological damage for rapid sealing. The laticifer ducts of
M. zehntneri evidence a mixed pattern of development. Cell
fusion occurs at the same time as the apices of the laticifer
branches and their projections elongate through intercel-
lular spaces. This rather unusual type of development has
been reported in other species of the family (Canaveze and
Machado 2016; Canaveze et al. 2018). The primary latic-
ifers of Hevea brasiliense, for example, develop by intrusive
growth, while its secondary laticifers develop by cell fusion
mediated by cell wall enzymes, such as expansins and pectin
esterases (Tan et al. 2017).
The observation of acuminate ends in laticifers usually
indicates intrusive growth, although acute apices have been
mistakenly identified in anastomosed laticifers in oblique
histological sections, making observational caution nec-
essary (Gonçalves et al. 2018; Salomé et al. 2022; Souza
et al. 2021). Ultrastructural analyses can greatly assist in the
to October. c Thicknesses of the cambial band and the secondary
phloem, with higher values in the dry season, between March and
May
correct characterization of the structural types of laticifers
through the detailed documentation of dissolution processes
and the intrusive elongation of the cell wall. The dissolu-
tion of the cell wall can occur in a centrifugal manner, pre-
ceded by a concentration of plasmodesmata associated with
the endoplasmic reticulum that supply lytic enzymes that
degrade cell wall components (Gama et al. 2017; Gonçalves
et al. 2018; Salomé et al. 2022; Souza et al. 2021). That elon-
gation and penetration of walls through the median lamella
may not be perceptible using light microscopy (Canaveze
and Machado 2016; Salomé et al 2022). Immunocytochemi-
cal studies using electron microscopy, however, can identify
the locations of cell wall loosening enzymes in the median
lamella of adjacent precursor cells (Serpe et  al. 2002;
Canaveze et al. 2018; Marinho and Teixeira 2019).
13
19 
Page 14 of 20
Fig. 9  Cambial activity in the stem of Marsdenia zehntneri Fontella
(Apocynaceae) as a function of climatic seasonality. a, b Cambial
activity during the dry period, with xylem production in March and
Fig. 10  Vegetative and reproductive phenologies of Marsdenia zehnt-
neri Fontella (Apocynaceae). Production of new leaves and floral
buds during the period when day length, rainfall, and temperature are
increasing. Fruit ripening during the dry period (See Fig. 8a, b)
13
Planta (2023) 257:19
April, and phloem production in May. c, d Vascular cambium inactive
in the rainy season. co cortex, pe periderm, ph secondary phloem, xy
secondary xylem
How do ultrastructural aspects relate to the latex
secretory process?
The processes of terpenoid and protein synthesis, which are
the major components of M. zehntneri latex, are related to
the presence of plastids, endoplasmic reticulum, and ribo-
somes. Latex secretion occurs during deep ultrastructural
alterations of laticifer precursor cells, which can only be
observed early, and for a short time, in the apical stem mer-
istem (Serpe et al. 2002; Gonçalves et al. 2018; Salomé
et al. 2022; Souza et al. 2021). The observation of oleoplasts
(vesicles containing electron-dense contents released from
the smooth endoplasmic reticulum) as well as free terpene
droplets in the cytosol are associated with the accumula-
tions of the terpenes found in latex. These droplets will be
engulfed by the central vacuole containing the hydrophilic
fraction of the latex—thus forming an emulsion (Nakazawa
et al. 2013; Naidoo et al. 2020; Gonçalves et al. 2018; Gama
et al. 2017; Ramos et al. 2019; Souza et al. 2021). Those ter-
pene droplets correspond to the rubber particles in the latex
Planta (2023) 257:19 Page 15 of 20  19
of the rubber tree H. brasililensis (Euphorbiaceae) (Sando
et al. 2009).
The hydrophilic content of M. zehntneri latex is com-
posed of proteins, acidic polysaccharides, alkaloids, and
phenolic compounds (Naidoo et al. 2020; Campos et al.
2021). Latex protein synthesis is associated with the pres-
ence of rough endoplasmic reticulum, free polysomes, and
protein bodies in the protoplast of laticiferous precursor cells
(Naidoo et al. 2020; Gonçalves et al. 2018; Ramos et al.
2019; Souza et al. 2021). Some of those proteins have the
function of clotting the latex when it is exuded in response
to injuries to plant tissue, while others act to defend against
biological agents (Konno 2011; Freitas et al. 2016, 2020).
Catalytic enzymes have been reported to participate in the
collapse of the portion of the cytoplasm that will compose
the latex during laticifer ontogenesis (Zhang et al. 2018;
Marinho and Teixeira 2019). Acidic polysaccharides are
commonly encountered in secretory structures (Mercadante-
Simões and Paiva 2013) and are associated with the occur-
rence of large populations of dictyosomes in precursor cells.
Vesicles released from dictyosomes fuse to the central vacu-
ole and discharge their contents into it, making up part of
the hydrophilic latex fraction (Gama et al. 2017; Gonçalves
et al. 2018; Souza et al. 2021).
Alkaloids are delivered to the laticifer from the phloem,
and phenolic compounds synthesized in the cytosol of pre-
cursor cells are likewise be added to the vacuole contents to
compose the latex (Almeida et al. 2012; Naidoo et al. 2020;
Onoyovwe et al. 2013; Uzaki et al. 2022). Bulky nuclei and
nucleoli as well as numerous mitochondria are responsible
for enzyme production and for supplying the energy neces-
sary for the synthesis of plant secretions. Those processes
also occur in the laticifers themselves (Marinho and Teixeira
2019; Gonçalves et al. 2018; Teixeira et al. 2020; Souza
et al. 2021). The relationship between the presence of certain
cellular components and the synthesis of latex compounds is
supported by histochemical studies that, in turn, can guide
further chemical analyses (Nakazawa et al. 2013; Gonçalves
et al. 2018; Naidoo et al. 2020; Souza et al. 2021; Salomé
et al. 2022; Lopes et al. 2009).
What are the relationships among biological
activity, ecological function, and the chemical
nature of latex?
The chemical compounds present in latex are related to the
adaptation of a plant species to its natural environment and
its medicinal potential. In general, latex is white, opaque,
sticky, and coagulates in contact with air; its viscosity is due
to high concentrations of rubber molecules (Agrawal and
Konno 2009). When centrifuged, the latex separates into
a light upper fraction rich in rubber, an intermediate frac-
tion represented by the laticifer cytosol, and a heavy lower
fraction rich in luteids (protein-rich organelles) (Habib and
Ismail 2020). The compound present in the greatest amounts
is cis-1,4-polyisoprene, which is formed through a sequential
condensation of isopentenyl diphosphate (isoprene) mole-
cules synthesized in the mevalonate pathway in the cyto-
sol and plastids of the laticifer precursor cells (Agrawal and
Konno 2009; Naidoo et al. 2020; Habib and Ismail 2020).
With the exudation of the latex and the subsequent decrease
in the turgor pressure of the intact laticifer, the luteids rup-
ture and their constituent proteins are trapped between the
polyisoprene micelles to form a clot. This protein network
allows for a quick occlusion of the wound and forms a plug
that stops further exudation (Shi et al. 2019; Ramos et al.
2019; Habib and Ismail 2020).
Several latex chemical compounds show biotechnological
potentials for drug development and have been prospected
based on reports of traditional medicinal uses of latex-
secreting species (Gonçalves et al. 2018; Souza et al. 2021;
Petricevich and Abarca-Vargas 2019; Salomé-Abarca et al.
2019). Some species of Marsdenia are widely used in the
traditional Chinese medicine (Chen et al. 2022). Among
those species, M. tenassissima stands out for its antitumor
activity (Lin et al. 2022) and M. globulifera as an invig-
orator of stomach functions (Sheng-Xiang et  al. 1993).
Many of these effects are attributed to the high concentra-
tions of terpenoids found in latex (Alqahtani et al. 2013;
Ramachandran and Prasad 2008; Mishra et al. 2010; Tijjani
et al. 2012; Petricevich and Abarca-Vargas 2019). Ursolic
acid has a protective effect against ultraviolet-B radiation-
induced cytotoxicity (Ramachandran and Prasad 2008), lup-
20(29)-en-24-oic acid has antimicrobial effects (Mishra et al.
2010), betulin has anticancer and anti-inflammatory effects
(Tijani et al. 2012; Ali-Seyed et al. 2016; Algahtani et al.
2013), and b-amyrin evidences multiple biological activi-
ties (Petricevich and Abraca-Vargas 2019). Polyisoprenes
are among the most important natural polymers produced
by plants and serve as raw materials for human needs. Rub-
ber, for example, is produced from the latex harvested from
H. brasililense, and is a very important commodity from
tropical regions (Nakazawa et al. 2013). The most frequent
proteins found in latex are proteases, especially chitinases
with antifungal properties. Other proteins evidence various
biological activities, such as peptidases with cardiotoxic and
hepatotoxic effects, lecithin that binds to carbohydrates and
favors rubber coagulation, osmotin with antimycotic action,
and thaumatin that is reported to be the sweetest natural
substance known (3000 times sweeter than sugar) (Agrawal
and Konno 2009; Freitas et al. 2016).
Latex has been of great evolutionary importance to
plants, because it favors their adaptation to many environ-
ments, especially tropical regions, where they have more fre-
quent interactions with microorganisms and insects (Farrel
et al. 1991; Konno 2011). Polyisoprene and protein clots seal
13
19 
Page 16 of 20
wounds and help prevent infection by microorganisms. Chi-
tinases destabilize the spore envelopes of phytopathogenic
fungi and their cell walls, thus affecting their cytoplasmic
contents. Peptidases cause damage to biological structures
through catalytic actions similar to the effects of snake
venom. Coagulated latex will also clog the oral apparatus
of herbivorous insects as well as reduce nutrient assimilation
by adhering to their periotrophic layer (Ramos et al. 2019;
Salomé-Abarca et al. 2019).
What are the effects of climate seasonality
on laticifer formation and phenology?
The development of laticifers in the primary structure occurs
during the rainy season, with the emission of leaves and
flowers. Laticifer development in secondary phloem occurs
during the dry period, during fruit ripening. The formation
of secondary phloem in M. zehntneri during the dry period
differs from the cambial activity patterns of other latex-
producing species of Apocynaceae growing in the same
study area (Fig. 11). The phloem of H. speciosa develops
at the end of the rainy season (Souza et al. 2021), but in
C. procera, it is produced during the entire rainy season
(Salomé et al. 2022). These different developmental pat-
terns are related to the water-use strategies of those species.
M. zehntneri grows in rocky soils that accumulate water in
crevices, thus prolonging the period of water availability.
H. speciosa, on the other hand, grows in deep soils with
low water retention capacities; C. procera grows in deep
soils and has several adaptations against water losses in
arid environments. The developmental patterns of species
occurring in the same environment can vary considerably, as
meristematic activity and phenological patterns are the result
Fig. 11  Cambial activity patterns and secondary phloem and laticifers formation in Marsdenia zehntneri Fontella, Hancornia speciosa Gomes
(Apocynaceae), and Calotrops procera (Ailton) W.T. Ailton (Apocynaceae), latescent species of Apocynaceae, occurring in the same study site
13
Planta (2023) 257:19
of a variety of selective pressures such as seasonal climatic
variations, nutrient and water availability in the soil, and the
presence of pollinators, predators, and seed dispersers that
allow (or discourage) the establishment of certain species in
the environment (Fenner 1998).
Precipitation and temperature have been described as the
main triggers of cambial activity and phenophases in plants
growing in tropical environments (Reich and Borchert 1984;
Wright and Cornejo 1990; Wright 1991; Van Schaik et al.
1993; Borchert 1999; Marcati et al. 2006). Day length seems
to affect cambial activity more than rainfall or temperature
among species growing in the Brazilian Cerrado (Marcati
et al. 2016; Lara et al. 2017), and its action is mediated by
auxin and abscisic acid (Fischer et al. 2019). Flowering was
observed to occur in species of Marsdenia in response to
increasing day length, even in the dry season (Juárez-Jaimes
and Maria Ángeles 2013). Cambial activity was stimulated
in Cordiera concolor (Rubiaceae) by increasing day length
after long-term dormancy, while cambial dormancy was
anticipated by water deficits (Lara and Marcati 2016; Lara
et al. 2017). Day length and temperature determined bud
opening and the beginning of cambial activity in Kielmeyera
grandiflora (Calophyllaceae), with the peak of vascular tis-
sue development occurring when the leaves are mature and
rainfall abundant (Bosio et al. 2016).
It is important to understand the integrated actions
of multiple factors in the development of phloem, where
laticifers are found. The genes involved in defenses against
biotic stress in the rubber tree H. brasiliensis were found
to be related to the activities of primary laticifers, while
the genes involved in managing abiotic stress were found to
be related to the activities of secondary laticifers—indicat-
ing that those two types of laticifer are functionally distinct
Planta (2023) 257:19 Page 17 of 20  19
(Tan et al. 2017). Reductions in latex flow were recorded
in H. brasiliense during leaf emission, which is a relevant
observation considering the importance of latex extraction
for producing natural rubber (Zhai and Xu 2022). The devel-
opment of laticifers in the primary structure of M. zehntneri
occurs in the rainy season, during the emission of leaves and
flowers—therefore providing protection against the herbi-
vores and phytopathogens that occur in abundance under
humid conditions. The formation of secondary phloem and
secondary laticifers during the dry season does not compete
with the energy requirements of leaf, flower, and fruit devel-
opment during the rainy season.
Conclusion
The laticifer network of M. zehntneri permeates the entire
primary and secondary body of the plant, constituting a
broad distribution and delivery system for defensive com-
pounds. Its peculiar type of anastomosed laticifer with intru-
sive growth constitutes a model for studies of the dynamics
of cell wall resorption and elongation among precursor cells.
The processes of synthesis of terpenoids and proteins, the
major components of latex, are related to the presence of
plastids, endoplasmic reticulum, and ribosomes, and were
supported here by histochemical studies and specific chemi-
cal analyses. Methyl 2,3-dihydroxy-ursan-23-oate, methyl
3-hydroxy-ursan-23-oate, and betulin account for more than
57% of the substances identified in the látex. Those com-
pounds are also found in other species of the genus Marsde-
nia, and may constitute taxonomic markers. The recognized
biological properties of those compounds contribute to the
survival of that species in the harsh environments of karst
outcrops in the Brazilian Cerrado. The development of pri-
mary laticifers in elongating shoot apices is triggered by
increasing day length, while the production of secondary
laticifers from cambial derivatives of the phloem is triggered
by decreasing temperatures and precipitation. The primary
structure laticifers protect the elongating plant during the
rainy season. The cambial activity and the increases in stem
thickness during the dry season do not compete with vegeta-
tive expansion and reproduction during the rainy season. It is
important to understand the joint actions of multiple factors
in the development of the phloem, where the laticifers are
found, in terms of the production and harvesting of latex.
The wide distribution of laticifers, the seasonal pattern of
latex secretion, and its composition contribute to the adapta-
tion of M. zehntneri to its natural environment.
Author contribution statement  MOM-S conceived and
designed the research and performed the ultrastructural
analyses. LMR analyzed the quantitative data. ISP Azevedo
conducted the fieldwork and analyzed the environmental
data. HKOM, WSSP, MGFF performed the anatomical, his-
tochemical, morphometric, and micro-morphometric analy-
ses. LPD, GFS and MGA performed the chemical analyses.
All authors contributed to writing the text.
Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00425-0​ 22-0​ 4050-7.
Acknowledgements  This work was supported by the Fundação de
Amparo à Pesquisa do Estado de Minas Gerais—FAPEMIG (CRA-
PPM-00544-18) and by the Conselho Nacional de Desenvolvimento
Científico e Tecnológico—CNPq (423340/2018-2, 441440/2016-9 and
441583/2020-2—Long-term Ecological Research Network—PELD-
VERE). The authors thank to CNPq for the Research Productivity
Grants awarded to MO Mercadante-Simões, (303806/2019-2) and LM
Ribeiro (308337/2021-2); the Microscopy Center, Federal University of
Minas Gerais, for ultrastructural analyses; the High Resolution Nuclear
Magnetic Resonance Laboratory, Chemistry Department, Federal
University of Minas Gerais for the chemical analyses; and Alysson
Rocha Pereira and Rúbia Santos Fonseca for their support during the
fieldwork.
Data availability  All data generated or analyzed during this study are
included in this published article and its supplementary information
files.
Declarations 
Conflict of interest  The authors have no competing interests to declare
that are relevant to the content of this article.
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