Download Induction of wound-periderm-like tissue in

Annals of Botany 116: 763–769, 2015
doi:10.1093/aob/mcv129, available online at www.aob.oxfordjournals.org
Induction of wound-periderm-like tissue in Kalanchoe pinnata (Lam.) Pers.
(Crassulaceae) leaves as a defence response to high UV-B radiation levels
Luana Beatriz dos Santos Nascimento1, Nattacha dos Santos Moreira1, Marcos Vinı́cius Leal-Costa2,
Sônia Soares Costa3 and Eliana Schwartz Tavares1,*
1
Plant Anatomy Laboratory, Botanical Department, Biology Institute, Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil, 2Federal Institute of Education, Science and Technology, Campos dos Goytacazes, Brazil and
3
Chemistry of Natural Bioactive Products Laboratory, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
*For correspondence. E-mail [email protected]
Received: 13 May 2015 Returned for revision: 29 June 2015 Accepted: 17 July 2015 Published electronically: 7 September 2015
Background and Aims UV-B radiation can be stressful for plants and cause morphological and biochemical
changes. Kalanchoe pinnata is a CAM leaf-succulent species distributed in hot and dry regions, and is rich in flavonoids, which are considered to be protective against UV-B radiation. This study aims to verify if K. pinnata has
morphological or anatomical responses as a strategy in response to high UV-B levels.
Methods Kalanchoe pinnata plants of the same age were grown under white light (control) or white light plus
supplemental UV-B radiation (5 h d–1). The plants were treated with the same photoperiod, photosynthetically active
radiation, temperature and daily watering system. Fragments of the middle third of the leaf blade and petiole were
dehydrated and then embedded in historesin and sectioned in a rotary microtome. Sections were stained with toluiR
. Microchemical analyses by optical microscopy were performed on fresh
dine blue O and mounted in EntellanV
material with Sudan III, Sudan IV and phloroglucinol, and analysed using fluorescence microscopy.
Key Results Supplemental UV-B radiation caused leaf curling and the formation of brown areas on the leaves.
These brown areas developed into a protective tissue on the adaxial side of the leaf, but only in directly exposed regions. Anatomically, this protective tissue was similar to a wound-periderm, with outer layer cell walls impregnated
with suberin and lignin.
Conclusions This is the first report of wound-periderm formation in leaves in response to UV-B radiation. This
protective tissue could be important for the survival of the species in desert regions under high UV-B stress
conditions.
Key words: Kalanchoe pinnata, Crassulaceae, leaf anatomy, fluorescence microscopy, radiation responses, UV-B
radiation, wound-periderm.
INTRODUCTION
A well-studied environmental stress of plant life is UV-B radiation, mainly due to the increased incidence of this radiation on
the surface of the Earth, because of the increasing depletion of
the ozone layer (Jansen et al., 1998; Ueda and Nakamura,
2011; Reboredo and Lidon, 2012). UV-B can promote morphological and biochemical changes in plants (Jansen et al., 1998;
Jenkins et al., 2014). It can act on DNA, promoting the formation of pyrimidine dimers, increase the generation of ROS (reactive oxygen species) and it can affect plant growth and
development (Jansen et al., 1998, 2012). On the other hand, recent discoveries concerning the positive effects of UV-B radiation and its responses, even with low doses, have shifted the
perception of this radiation from a stress to a signal (Jansen and
Bornman, 2012).
Morphological responses to UV-B can include leaf curling,
axillary branching, an increase in the root:stem ratio, inhibition
in shoot elongation, and an increase of the leaf area and thickness (Jansen, 2002; Robson et al., 2015). At the cellular level,
UV-B can provoke alterations in cell division, elongation and/
or differentiation (Jansen, 2002; Robson et al., 2015).
Kalanchoe pinnata (Lamarck) Persoon (syn.¼Bryophyllum
pinnatum, B. calycinum) is a member of the Crassulaceae family. The species is commonly distributed in tropical regions
(Allorge-Boiteau, 1996; Judd et al., 2009). Crassulacean acid
metabolism (CAM) and succulent leaves allow its acclimation
to environmental factors such as periodic drought and hot days
(Winter, 1985; Judd et al., 2009). In addition, the leaves of this
species are rich in flavonoids (Costa et al., 2008), substances
that are considered protective against UV-B radiation (Agati
et al., 2013), and that also play a role in many of the proven biological activities of this species (Costa et al., 2008). In a previous study, we verified the qualitative and quantitative changes
in the phenolic and flavonoid production of K. pinnata leaf extracts, induced by supplemental UV-B radiation (Nascimento
et al., 2015). This study aimed to verify whether, besides flavonoid production, this species has morphological or anatomical
responses as a strategy to deal with high UV-B levels.
C The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.
V
All rights reserved. For Permissions, please email: [email protected]
Nascimento et al. — Wound-periderm-like tissue induced by UV-B radiation
764
TABLE 1. Light treatment specifications
Treatment
White
lamps
UV-B
lamps
Total PAR
(mol m–2 s–1)
UV-B
supplementation
(5 h d–1, W m2)
W
W þ UVB
þ
þ
–
þ
200–400*
200–400*
–
4–15*
W, white light (control); W þ UVB, white light þ supplemental UV-B radiation treatment.
*Depending on the plant position with respect to the light source.
Photoperiod: 115 h light.
MATERIALS AND METHODS
Plant materials and growing conditions
Thirty plants produced vegetatively from three different
Kalanchoe pinnata (RFA37.525, RFA39.958 and RFA39.963)
matrix plants were planted in individual plastic pots (18 cm diameter and 15 cm high) filled with a commercial soil mixture
R
(NutriplanV
). Prior to the UV-B treatment, these plants were
grown on the campus of the Universidade Federal do Rio de
Janeiro, under sunlight, with photosynthetically active radiation
(PAR) varying from 400 to 800 mol m–2 s–1 and the same watering system. The PAR was measured with a PAR sensor coupled
to an FMS2 Hansatech fluorometer (Hansatech Instruments
Ltd, King’s Lynn, UK). After 6 months, these plants of
the same age were acclimatized in controlled-environment
growth chambers for 2 weeks under the same PAR (200–
400 mol m–2 s–1, depending on plant position), temperature
(32 6 4 C), manual irrigation (100 mL d–1) and photoperiod
(115 h of light). Plants were rotated every day, to minimize the
effects of positional PAR. These basic growing conditions remained the same throughout the experiment.
Light treatments
After acclimation, the plants were randomly allocated to two
different light treatments: white light (W; control) and white
light plus supplemental UV-B radiation for 5 h d–1 (from
1000 h to 1500 h; W þ UVB) (Table 1). Six 59 W daylight
fluorescent lamps (Golden, São Paulo, SP, Brazil) were used as
a source of white light for both treatments (W and W þ UVB);
and one tubular 20 W broadband UV-B lamp (290–320 nm;
UV-B Medical Phillips TL UV-B G13 T12) was used as a
source of supplemental UV-B radiation, only for the W þ UVB
treatment.
The spectral distribution of the daylight fluorescent lamp radiation was measured with a QM1 spectrofluorometer (PTI
Inc., Lawrenceville, NY, USA) (Nascimento et al., 2013). The
spectral distribution of the UV-B lamp was provided by the
manufacturer.
The W þ UVB treatment had a mean irradiance of 4–
15 W m–2 (72–200 kJ m–2 daily dose) of UV-B radiation (depending on the plant position), as measured with a VLX-3 W
radiometer (with a 312 nm flat sensor, positioned horizontally
during the measurements). The corresponding mean biologically effective UV-B irradiance (UVBBE), weighted using the
generalized plant action spectrum normalized at 300 nm
(Cadwell, 1971; Aphalo et al., 2012), was 027–102 W m–2
(486–1836 kJ m–2 daily dose). The distance between the plants
and the lamp was 20 cm, and the pots were rotated each day on
each bench, to minimize edge and position effects.
Collecting and leaf anatomy
Anatomical analyses were performed on fully developed
simple leaves (third node) from both treatments (W and
W þ UVB). The first leaf collection started prior to the first
UV-B supplementation (time zero – T0). Leaves of the plants
from the W and W þ UVB treatments were collected daily and
fixed in FAA70 (Johansen, 1940) until the 11th day, and after
that they were collected every 4 d, i.e. on the 15th, 19th and
23rd day (T15, T19 and T23, respectively). Leaf fragments
R
were dehydrated and then embedded in Leica HistoresinV
and
sectioned (10 m) in a Spencer rotary microtome. Cross-sections
were taken from the middle region of the petiole and in the
middle third of the leaf blade. The sections were stained with
Toluidine blue O (O’Brien et al., 1965) and mounted in
R
EntellanV
. Microchemical tests were performed on fresh material: Sudan III and IV to reveal lipids (Sass, 1951) and phloroglucinol to reveal lignin (Johansen, 1940). This fresh material
was analysed by optical and fluorescence microscopy. For optical microscopy, an Olympus CH30 light microscope was used
and the photographs were taken with an attached Olympus PMC35B camera.
For fluorescence, a Leica DMLB microscope was used.
Sudan IV was used in conjunction with a fluorescence I3 filter
(blue light emission, bandpass 450–490 nm and 515 nm barrier
filter) (Rittinger et al., 1987).
Leaf thickness measurements were made on cross-sections of
leaves collected from three plants grown for 23 d under control
and UV-B treatment. For each region and treatment, 15 meaR
surements were taken on a LabomedV
microscope equipped
with a digital camera and computer, used in conjunction with
R
Pixel ProV
Image Analysis Software.
Statistical analysis was conducted with GraphPad Instat 3.0
R
for WindowsV
. Data were analysed with Student’s t-test
(P < 00001).
RESULTS AND DISCUSSION
Morphological differences between W and W 1 UVB plants
Supplemental UV-B radiation induced morphological changes
in the K. pinnata leaves. At the beginning of the experiment
(T0), plants were morphologically identical.
However, as of the second day of the experiment (T2), the
leaves of the W þ UVB plants showed leaf curling in the direction of the adaxial side, mainly in the first and second node
leaves (Fig. 1A). This photomorphogenetic response to the radiation has been observed in several other species (Barnes et al.,
1996; Jansen et al., 1998; Zuk-Golaszewska et al., 2003;
Boeger and Poulson, 2006). The leaf curling can decrease the
leaf area exposed to the radiation and is due to a reduction in
cell wall expansion by cells on the adaxial side, compared with
those on the abaxial side (Wilson and Greenberg, 1993; Boeger
and Poulson, 2006). Auxins appear to play a fundamental role
Nascimento et al. — Wound-periderm-like tissue induced by UV-B radiation
A
B
C
D
E
F
765
FIG. 1. Morphological changes in K. pinnata leaves induced by supplemental UV-B radiation (A–E). (A) Plant from T2: leaf curling in the direction of the adaxial
side (arrows). (B) Leaf from a T2 plant: brown areas on the adaxial side of the leaf blade were detected (arrows). (C) Plant from T5: a brown film (protective tissue)
on the adaxial surface, only in areas directly exposed to UV-B (arrows). (D) Detail of the brown film formed in response to UV-B radiation on the leaf blade and petiole. (E) Leaf from a T23 plant showing that the leaf margin buds were alive (arrows). (F) Control T23 plant: no changes in plant morphology were seen. The purple
colour in the petiole and leaf margin is common in K. pinnata, and has no relationship to the brown areas formed in response to UV-B radiation.
in this process. Indeed, this class of hormone has been shown to
be the main substance that induces the common morphological
changes in response to UV-B in plants (Jansen, 2002; Hectors
et al., 2012).
Brown areas were noticed on the adaxial surface of the
W þ UVB leaves, especially those from the first and second
nodes, but only in regions directly exposed to UV-B (Fig. 1B).
During the UV-B exposure, these brown areas developed into a
‘brown film’ (Fig. 1C) on the leaf blade and petiole (Fig. 1D).
Studies with field crops under UV-B radiation showed the occurrence of bronze or brown spots on leaves (Kakani et al.,
2003), and these brown spots developed necrosis and chlorosis,
and the leaves suffered desiccation and senescence. However,
in these previous works there are no mentions of the foliar anatomy of these areas (Kakani et al., 2003). In our work, the
K. pinnata leaves did not develop necrosis or chlorosis.
The apical bud and leaf margin buds (Fig. 1E, arrows) remained alive until the end of the experiment. Morphological
changes were not observed on the abaxial side of the W þ UVB
leaves or in leaves from the W treatment through till the end of
the experiment (T23) (Fig. 1F).
Leaf anatomy
At time zero (T0) the anatomy of the petiole (Fig. 2A) and
the leaf blade (Fig. 4A) were similar in both W and W þ UVB
plants, with no differences from those previously described by
Moreira et al. (2012).
On the second day of the experiment (T2), petioles of the
W þ UVB plants showed periclinal cell division (Fig. 2B, star)
and loss of periclinal walls (Fig. 2B, asterisk) in some subepidermal regions of the adaxial surface. In addition, in some
regions on the adaxial side there was a loss of epidermis
(Fig. 2B, arrows). Anticlinal and periclinal divisions occupied
more continuous regions in the course of the UV-B exposure
Nascimento et al. — Wound-periderm-like tissue induced by UV-B radiation
766
A
B
100 µm
C
100 µm
D
50 µm
50 µm
E
F
200 µm
100 µm
FIG. 2. Transversal sections from the petiole of K. pinnata cultivated under supplemental UV-B radiation. (A) On day zero (T0): no changes in the petiole anatomy.
(B) On the second day of UV-B exposure (T2): periclinal cell division (star) and loss of periclinal cell walls (asterisk) in the sub-epidermal regions; loss of epidermis
on the adaxial side (arrows). (C) On the fifth day of UV-B exposure (T5): anticlinal and periclinal cell divisions, occupying more continuous regions; collapsed epidermis on the adaxial side (arrow). (D) On the tenth day of UV-B exposure (T10): detail of wound-periderm tissue on the adaxial side, with three distinct cell types:
meristematic cells with pronounced nuclei (ellipse); externally from these cells, cells with thick walls impregnated with lipid and lignin; and internally from the
meristematic cells, more differentiated daughter cells (asterisk). (E) On the 19th day of UV-B exposure (T19): external layers of protective tissue (wound-periderm)
broken (arrow). (F) On the 23rd day of UV-B exposure (T23): lenticel-like structures (arrow) on the petiole adaxial side.
from the fifth day onwards (T5) (Fig. 2C). At this time, the epidermis collapsed (Fig. 2C, arrows). As of the tenth day of the
UV-B exposure (T10) these divisions began to form a woundperiderm (Fig. 2D). Below the layers of the pre-existing cells
there are meristematic cell layers (phellogen) with rectangular,
thin cells with thin walls, and the nuclei are clearly seen
(Fig. 2D, ellipse). Above this layer, the recent cells formed by
meristematic action, between the meristem and pre-existing
cells, showed stronger suberin impregnation (phellem), as evidenced by Sudan IV observed under the fluorescence microscope using an I3 filter (Fig. 3A, arrow). Combining
fluorescence microscopy analysis with Sudan IV, it is possible
to observe the suberized cells in orange to red (Rittinger et al.,
1987). Below the meristematic layer, there are more differentiated parenchymatic cells (Fig. 2D, asterisk), the phelloderm.
The microchemical tests showed negative results for suberin
and lignin impregnation in phelloderm cell walls. The preexisting cells of the more external layers differentiated thick
walls, with suberin and lignin, evidenced by Sudan III and IV
reagents and phloroglucinol.
As of the 19th day of the experiment (T19), the external layer
of the pre-existing lignified cells began to break up (Fig. 2E,
arrow) and, as of the 23rd day (T23), lenticel-like structures
could be seen (Fig. 2F, arrow).
In the leaf blade, similar to the petiole, there was a loss and
collapse of the epidermis on the adaxial side of the leaves on
the second day (T2) of the experiment (Fig. 4B, arrow).
However, the periclinal cell divisions in the adaxial subepidermal cell layers only occurred as of the fifth day of the
experiment (T5) (Fig. 4C, stars). Also, besides the loss of
Nascimento et al. — Wound-periderm-like tissue induced by UV-B radiation
A
767
B
50 µm
200 µm
FIG. 3. Microchemical tests of transversal sections from the petiole and leaf blade of K. pinnata cultivated during 23 d under supplemental UV-B radiation. (A) Test
with Sudan IV on the petiole under a fluorescence microscope using an I3 band pass filter: stronger red colour in the walls of cells immediately above the meristematic tissue (arrow) reveals the stronger suberin impregnation when compared with cells above them (arrowhead). (B) Test with phloroglucionol reagent on a leaf
blade observed under the optical microscope exibiting a positive result for lignin impregnation in cell walls of the external cell layers.
chloroplasts on the adaxial side, there was a loss of epidermis
papillae (Fig. 4C, arrow). As of the sixth day of the experiment
(T6), anticlinal and periclinal divisions occupied more continuous regions and, as of the tenth day (T10), the wound-periderm
began to be formed (Fig. 4D). The tissue constitution of the leaf
blade (Fig. 4E) is similar to that of the petiole, with phellogen
(Fig. 4E, ellipse), phellem (Fig. 3B, star) and phelloderm
(Fig. 3B, asterisk). In all the newly formed tissue, rows of radial
cells are produced by anticlinal divisions (Fig. 3B).
Towards the end of the experiment, the protective tissue had
expanded (T23, Fig. 4F). Similarly for the petiole, the external
layers of tissue broke down, but only at the end of the experiment
(T23). At this time, a significant increase in leaf thickness could
be seen (Table 2), mainly due to the production of phelloderm.
The petiole and leaf blade anatomy of the control plants (W)
did not change throughout the experiment, and it was similar to
what was described for the species in our previous study
(Moreira et al., 2012) and similar to the leaf anatomy at time
zero (T0) for the UV-B plants (Figs 2A and 4A).
The wound healing process in the leaf blade and petiole of
Bryophyllum calycinum (syn. ¼ Kalanchoe pinnata) was previously studied by Welch (1945). The author made cuts in the
leaves with a razor blade and noticed the production of suberized–lignified cells originated by the action of a meristematic
layer in the wounded regions.
Rittinger et al. (1987) studied the healing process caused by
wounding and infection in leaves of three different species
(Mallus domestica Borkh., Prunus cerasus L. and Hordeum
vulgare L.), but the authors did not observe wound-periderm
formation in any of the studied species. On the other hand, the
development of wound-periderm as a consequence of necrosis
provoked by mechanical or pathogenic agents in plant organs,
including leaves, has been known for a long time; such data can
be found in Küster’s book (Küster, 1916). In fact, wound-periderm can be formed in response to wounds, irrespective of their
nature (Ginzberg, 2008).
Wound-periderm formation is common in tubers and has already been described in response to c-radiation in potatoes
(Solanum tuberosum) (Thomas, 1982). The wound-periderm is
produced in two stages; the first stage, immediately after
wounding, is characterized by the generation of a suberized barrier referred to as a closing or sealing layer (Lulai, 2007). The
second stage is the formation of a meristematic cell layer,
which divides to produce suberized cells (Gizberg, 2008; Lulai
et al., 2014). In our study, as of the second day of the
experiment (T2) for the petiole and as of the fifth day (T5) for
the leaf blade, periclinal cell divisions were observed in the
sub-epidermal layers to form the phellogen, without a previous
suberization of the outer cell layers. So, the formation of this
wound-periderm in K. pinnata leaves seems to be different
from that in potatoes and other species that follow this pattern.
UV-B radiation caused damage to the leaf blade and petiole
epidermis [loss of epidermis and collapse of epidermal cells
(Figs 2B, C and 4B, C)] as of the initial days of exposure to the
radiation, and this could have triggered the wound-periderm
formation.
The formation of a wound-periderm induced by UV-B radiation has not yet been reported in the literature. Varied responses
to UV-B are related to different species genotypes and experimental conditions (Jansen, 2002; Xu and Sullivan, 2010;
Robson et al., 2015), making the analysis complex.
Reported anatomical changes induced by UV-B, in general,
include an increase in mesophyll, epidermis and wax cuticle
thicknesses (Boeger and Poulson, 2006; Fukuda et al., 2008;
Mörsky et al., 2013), and many of them act as protection
against radiation (Jansen et al., 1998; Jansen, 2002). These findings are consistent with ours, since we observed an increase in
leaf thickness.
Conclusions
Kalanchoe pinnata is a species that is well adapted to arid regions, mainly due to its CAM metabolism, succulent leaves
(Judd et al., 2009) and high phenolic composition. The woundperiderm-like tissue formed as a response to UV-B radiation
can act as an additional ‘tool’ for protection of the species
against radiation. The formation of such tissue in response to
the administration of high doses of UV-B seems coherent. In
conclusion, this study contributed to elucidate a leaf structural
response to UV-B radiation, not yet reported in the literature
and not common to this organ. This response is similar, but not
equal, to that described previously by Welch (1945), triggered
by cuts. The formation of wound-periderm-like tissue is probably an important acclimation response that K. pinnata has in
order to survive in regions which are hot, dry and with high
768
Nascimento et al. — Wound-periderm-like tissue induced by UV-B radiation
A
B
100 µm
C
100 µm
D
200 µm
100 µm
F
E
50 µm
200 µm
FIG. 4. Transversal sections from a K. pinnata leaf blade of the supplemental UV-B-radiated plants (A) On day zero (T0). (B) On the second day of UV-B exposure
(T2): collapse of epidermis (arrow). (C) On the fifth day of the experiment (T5): periclinal cell divisions in the adaxial sub-epidermal layers (stars); loss of adaxial
chloroplasts and epidermis papillae (arrow). (D) On the tenth day of UV-B exposure (T10): wound-periderm formed on the adaxial side. (E) On the tenth day of
UV-B exposure (T10): detail of wound-periderm with three distinct cell types; meristematic cells (ellipse), phellem cells and phelloderm cells. (F) On the 23rd day
of UV-B exposure (T23): wound-periderm had expanded significantly.
TABLE 2. Leaf thickness of K. pinnata plants grown for 23 d under
white light (control) and white light supplemented with UV-B
radiation
W
W þ UVB
Petiole
Leaf blade
316869 6 6527 mma
363664 6 11093 mma
79740 6 3464 mmb
11364 6 4063 mmb
W, white light (control); W þ UVB, white light þ supplemental UV-B radiation treatment.
Different letters in the same line indicate a statistical difference P < 00001,
n ¼ 15.
UV-B levels. Despite the absence of previous records of
wound-periderm formation in response to UV-B radiation, it
may be more widespread than we imagine. The brown areas
seen in plants submitted to high sunlight or UV-B exposure
should be analysed in order to verify their anatomical nature.
ACKNOWLEDGEMENTS
We thank Mr David Straker for language revision, and Ms
Claudia Lage for her contribution. This work was supported by
the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro
Nascimento et al. — Wound-periderm-like tissue induced by UV-B radiation
[FAPERJ; grant no. E-26/111.390/2013] and the Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico [CNPq;
grant no. 161598/2012-9].
LITERATURE CITED
Agati G, Brunetti C, Di Ferdinando M, Ferrini F, Pollastri S, Tattini M.
2013. Functional roles of flavonoids in photoprotection: new evidence, lessons from the past. Plant Physiology Biochemistry 72: 35–45.
Allorge-Boiteau L. 1996. Madagascar centre de speciation et d’origine du genre
Kalanchoe (Crassulaceae). In: WR Lourenço, ed. Biogéographie de
Madagascar. Paris: Éditions de l’ORSTOM, 137–145.
Aphalo PJ, Albert A, Björn LO, Mcleod A, Robson TJ, Rosenqvist E. 2012.
Beyond the visible: a handbook of best practice in plant UV photobiology.
Helsinki: University of Helsinki, Division of Plant Biology.
Barnes DJ, Percy KE, Paul ND, et al. 1996. The influence of UV-B radiation
on the physicochemical nature of tobacco (Nicotiana tabacum L.) leaf surfaces. Journal of Experimental Botany 47: 99–109.
Boeger MRT, Poulson M. 2006. Efeitos da radiação ultravioleta-B sobre a morfologia foliar de Arabidopsis thaliana (L.) Heynh. (Brassicaceae). Acta
Botanica Brasilica 20: 329–338.
Caldwell MM. 1971. Solar UV irradiation and the growth and development of
higher plants. In: AC Giese, ed. Photophysiology. New York: Academic
Press, 131177.
Costa SS, Muzitano MF, Camargo LMM, Coutinho MAS. 2008. Therapeutic
potential of Kalanchoe species: flavonoids and other secondary metabolites.
Natural Products Communication 3: 2151–2164.
Fukuda S, Satoh A, Kasahara H, Matsuyama H, Takeuchi Y. 2008. Effects
of ultraviolet-B irradiation on the cuticular wax of cucumber (Cucumis sativus) cotyledons. Journal of Plant Research 121: 179–189.
Gizberg I. 2008. Wound-periderm formation. In: A Schaller, ed. Induced plant
resistance to herbivory. Berlin: Springer, 131–146.
Hectors K, Oevelenb SV, Guiseza Y, Prinsenb E, Jansen MAK. 2012. The
phytohormone auxin is a component of the regulatory system that controls
UV-mediated accumulation of flavonoids and UV-induced morphogenesis.
Physiologia Plantarum 145: 594–603.
Jansen MAK. 2002. Ultraviolet-B radiation effects on plants: induction of morphogenic responses. Physiologia Plantarum 116: 429–429.
Jansen MAK, Bornman JF. 2012. UV-B radiation: from generic stressor to specific regulator. Physiologia Plantarum 145: 501–504.
Jansen MAK, Gaba V, Greenberg BM. 1998. Higher plants and UV-B radiation: balancing damage, repair and acclimation. Trends in Plant Science 3:
131–135.
Jansen MAK, Hideg E, Lidon FJC. 2012. UV-B radiation: when does the stressor cause stress? Emirates Journal of Food and Agriculture 24 (6): 1–3.
Jenkins GI. 2014. The UV-B photoreceptor UVR8: from structure to physiology. The Plant Cell 26: 21–37.
Johansen DA. 1940. Plant microtechnique. New York: McGraw-Hill Book
Company Inc.
Judd WS, Campbell CS, Kellogg EA, Stevens PF, Donoghue MJ, 2009.
Sistemática vegetal: um enfoque filogenético, 3rd edn. Porto Alegre: Artmed.
769
Kakani VG, Reddy KR, Zhao D, Sailaja K. 2003. Field crop responses to
ultraviolet-B radiation: a review. Agriculture and Forest Meteorology 120:
191–218.
Küster E. 1916. Pathologische Pflanzenanatomie: In ihren Grundzügen, 2nd
edn. Jena: G. Fischer.
Lulai EC. 2007. The canon of potato science: skin-set and wound-healing/suberization. Potato Research 50: 387–390.
Lulai EC, Neubauer JD, Suttle JC. 2014. Kinetics and localization of wound
induced DNA biosynthesis in potato tuber. Journal of Plant Physiology
171: 1571–1575.
Moreira NS, Nascimento LBS, Leal-Costa MV, Tavares ES. 2012.
Comparative anatomy of leaves of Kalanchoe pinnata and K. crenata in sun
and shade conditions, as a support for their identification. Brazilian Journal
of Pharmacognosy 22: 929–936.
Mörsky SK, Haapala JK, Rinnan R et al. 2013. Effects of elevated UV-B radiation on UV absorbing pigments and leaf anatomy of a sedge, Eriophorum
russeolum. Boreal Environment Research 18: 414–424.
Nascimento LBS, Leal-Costa MV, Menezes EA, et al. 2015. UltravioletB radiation effects on phenolic profile and flavonoid content of
Kalanchoe pinnata. Journal of Photochemistry and Photobiology B
148: 73–81.
O’Brien TP, Feder N, McCully ME. 1965. Polychromatic staining of plant cell
walls by toluidine blue O. Protoplasma 59: 368–373.
Reboredo F, Lidon FJC. 2012. UV-B radiation effects on terrestrial plants – a
perspective. Emirates Journal of Food and Agriculture 24: 502–509.
Rittinger PA, Biggs AR, Peirson PR. 1987. Histochemistry of lignin and suberin deposition in boundary layers formed after wounding in various plant
species and organs. Canadian Journal of Botany 65: 1886–1892.
Robson TM, Klem K, Urban O, Jansen MK. 2015. Re-interpreting plant morphological responses to UV-B radiation. Plant, Cell and Environment 38:
856–866.
Sass JE. 1951. Botanical microtechnique, 2nd edn. Ames, IA: The Iowa State
College Press.
Thomas P. 1982. Wound-induced suberization and periderm development in potato tubers as affected by temperature and gamma irradiation. Potato
Research 25: 155–164.
Ueda T, Nakamura C. 2011. Ultraviolet-defense mechanisms in higher plants.
Biotechnology & Biotechnological Equipment 25: 2177–2181.
Welch WB. 1945. Cicatrization in leaves of Bryophyllum calyclnum. Botanical
Gazette 107: 95–106.
Wilson MI, Greenberg BM. 1993. Specificity and photomorphogenic nature of
ultraviolet-B-induced cotyledon curling in Brassica napus L. Plant
Physiology 102: 671–677.
Winter K. 1985. Crassulacean acid metabolism. In: J Barber, NR Baker, eds.
Photosynthetic mechanisms and the environment. Amsterdam: Elsevier,
329–387.
Xu C, Sullivan JH. 2010. Reviewing the technical designs for experiments with ultraviolet B radiation and impact on photosynthesis, DNA
and secondary metabolism. Journal of Integrative Plant Biology 52:
377–387.
Zuk-Golaszewska K, Upadhyaya MK, Golaszewski J. 2003. The effect of
UV-B radiation on plant growth and development. Plant, Soil and
Environment 49: 135–140.