Lignification in Higher Plants

1. 10/11/2018
Department of Plant Biotechnology
1
WELCOME

2. THE CELL BIOLOGY OF
LIGNIFICATION IN HIGHER
PLANTS
MANJUNATH ,
R.
PALB 6281
10/11/2018Department of Plant Biotechnology
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3. CONTENTS
 INTRODUCTION.
LIGNIFIED CELL TYPES IN PLANTS.
CELL BIOLOGY OF LIGNIN MONOMER SYNTHESIS.
LIGNIN POLYMER FORMATION.
LIGNIN FORMATION DURING THE
DIFFERENTIATION OF SPECIFIC CELLS.
CONCLUSIONS.
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4. INTRODUCTION
• Lignin (Latin lignum ‘wood’) is a polyphenolic polymer deposited
directly in the cell wall of specialized cells.
• Mechanically weaker than cellulose, lignin adds a significant
reinforcement to cell wall, providing an additional tensile strength.
• Lignin is the second most abundant terrestrial biopolymer after
cellulose.
• Lignin forms in the spaces between the cellulose micro fibrils by
the oxidative coupling of free lignin monomers secreted directly
into the plant cell wall.
• The canonical lignin monomers, called monolignols, form H-
(hydroxyphenyl), G- (guaicyl) and S- (syringyl) units in the lignin
polymer.
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5. LIGNIFIED CELL TYPES IN PLANTS
• Cell wall lignification occurs during the differentiation of
distinct cell types, but also occurs in response to specific
environmental changes.
The cell types accumulating lignin during their differentiation
includes the following :-
• Tracheary elements (TEs).
• Sclerenchyma cells.
• Endodermal cell.
• Seed coat cells.
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6. TRACHEARY ELEMENTS (TES).
• These specialized cells are an important component of
the xylem: the vascular tissue responsible for the hydro-
mineral sap distribution and the mechanical resistance
of plants to gravity .
• TEs act as the plant sap-conducting cylinders and they
are formed by undergoing cell suicide to remove their
cell content and reinforcing their side walls with lateral
lignified secondary cell walls mainly composed of G-
units .
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7. SCLERENCHYMA CELLS
• These secondary cell wall-forming cells include fibres
and sclereids, found in many different plant tissues such
as xylem, phloem, epidermis and cortex in grasses and
cereals.
• The function of these cells is to strengthen the central
axis of plant organs mechanically against gravity,
mechanical disturbances and physical damage .
• The sclerenchyma fibres and sclereids have lignified
secondary cell walls mainly composed of S-units .
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8. ENDODERMAL CELLS
• Endodermal cells selectively allow passage of water and
solutes to the root vascular system by forming an
apoplastic barrier against the extracellular diffusion of
substances.
• This cell type constitutes the root endodermis which
delimits the root cortex from its vascular system.
• The Casparian strip :- Composed of lignin-like polymer
combined with suberin, tightly links the plasma
membranes and the apoplastic space between adjacent
endodermal cells .
• The polymer is composed of a mixture of G- and S-units
.
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9. SEED COAT CELLS
• During seed development, the ovule integuments differentiate
into several cell layers composed of different specialized cells
which will form the protective seed coat or testa.
• Seed coat cells develop heavily lignified secondary cell walls
to reinforce the outer surface of the seed mechanically and to
make it impermeable to liquids and gases.
• Seed coat lignins are different between angiosperm species
and are composed of a mixture of classic G- and S- units.
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10. • Lignin biosynthesis can also be triggered in responses to
various biotic and abiotic stresses.
• Such as :- Wounding,drought,low temperature, reduced
nutrient availability and CO2 or ozone exposure .
• These stress-induced lignin's generally impregnate the
primary cell wall of cells that are normally not lignified (i.e.
leaf epidermal or stem pith parenchyma cells) .
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11. LIGNIFIED CELL TYPES IN HIGHER
PLANTS.
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12. CELL BIOLOGY OF LIGNIN MONOMER
SYNTHESIS
• Lignin deposition depends on the cell type, the developmental stage
and the species.
• This spatial distribution is characterized by differences in time, amount,
size and monomeric composition of the lignin polymer .
• Lignin monomer precursor(s) derive from the aromatic amino acid
phenylalanine, tyrosine synthesized in the plastid, which is converted
into 4-hydroxyphenylpropene alcohols.
• The mobilization and storage of monolignols is regulated through
glycosylation/de glycosylation involving the enzymes UDP
glucosyltransferase (UGT).
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13. GENERAL PHENYLPROPANOID PATHWAY SHOWING
LIGNIN BIOSYNTHESIS IN ARABIDOPSIS THALIANA
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14. TISSUE-SPECIFIC EXPRESSION OF LIGNIN
MONOMER BIOSYNTHETIC GENES
• During plant development, lignin monomer biosynthetic genes
are expressed in all lignifying tissues (endodermis and xylem) as
well as in non-lignifying tissues such as the phloem parenchyma
and the epidermis.
• Lignin monomer biosynthetic genes are also expressed in the
non-lignified cambium and in xylem parenchyma cells
surrounding the lignifying TEs and fibres.
• The expression of the genes depends on the circadian/diurnal
cycle and responds to biotic and abiotic stresses.
(Moura et al.,
2010). 10/11/2018Department of Plant Biotechnology 14

15. SUB-CELLULAR LOCALIZATION OF
RELATED LIGNIN MONOMER
SYNTHESIS ENZYMES
• While the aromatic amino acid precursor initiating lignin monomer
synthesis derive from plastids, the enzymes implicated in lignin
monomer synthesis are cytoplasmic and associated with outer
surface of the endoplasmic reticulum (ER).
• The cytochrome P450 oxidoreductases is responsible for the
aromatic ring hydroxylation on the outer surface of the ER,
whereas all the other lignin monomer biosynthetic enzymes are
in the cytoplasm.
• Thus, the synthesis of monolignols occurs in specialized sub-
cellular, areas at the interface between the cytoplasm and the
outer surface of the ER.
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16. LIGNIN MONOMER TRANSPORT
MECHANISMS
• Although the synthesis of monolignols occurs within the cell
protoplast, lignin deposition is restricted to the cell walls.
Three types of transport mechanisms for the extracellular
secretion of lignin monomers :-
• Passive diffusion (PD).
• Vesicle-associated exocytosis.
• Active ATP dependent transport using ABC transporters /
proton coupled antiporters .
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17. • The passive diffusion is supported by in vitro observations of
the partitioning of lignin monomers by immobilized liposomes
and lipid bilayer discs.
• Vesicle-associated secretion of lignin monomers was initially
supported by the labelling of ER- and Golgi-derived vesicles in
xylem TEs when feeding with tritium-labelled phenylalanine
and subsequent auto radiographical imaging using electron
microscopy.
(Miao and
Liu,2010).
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18. STEM CROSS-SECTIONS OF (A) ARABIDOPSIS, (B) BRACHYPODIUM (C)
POPULUS .
BLUE ARROW: TRACHEARY ELEMENTS (TES), ORANGE ARROW : XYLEM
PARENCHYMA (XPS), RED ARROW: RAY PARENCHYMA(RPS) , PURPLE
ARROW: XYLARY FIBRES (XFS).
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19. LIGNIN MONOMER PRODUCTION –
AUTONOMOUS OR CO-OPERATIVE PROCESS
• Cell wall lignification could proceed through cell co-operation where
non-lignifying cells would provide the monomers and other
substrates/enzymes to the cell wall of actively lignifying cells.
• This mechanism is referred to as ‘The good neighbor hypothesis’ or
‘The non- cell autonomous process’.
• Co-operative lignin synthesis is clearly observed between TEs and
fibres in the xylem.
(Smith et al., 2013)
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20. • The Casparian strip of endodermal cells appears to depend
on cell autonomous lignification.
• Whereas xylem TEs appear to rely on a co-operative
lignification with neighbouring xylem TE precursors and the
surrounding parenchyma cells .
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21. LIGNIN POLYMER FORMATION
• The formation of the lignin polymer occurs directly in the cell
wall by the oxidative polymerization of secreted lignin
monomers.
• Different linkages of the polymer including both condensed
C–C linkages and non-condensed C–O–C ether linkages.
The monolignol radicals are produced enzymatically by two
kinds of phenol-oxido reductase enzymes :-
• O2- dependent Laccases .
• H2O2- dependent peroxidases.
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22. LACCASE- CATALYZED LIGNIN
POLYMER FORMATION
• Laccases do not appear to have specifically evolved to enable
plant lignin polymer formation and present a high degree of
structural conservation between bacteria, fungi and plants.
• Laccases are co-regulated with lignin monomer biosynthesis
genes, with secondary cell wall-forming genes and expressed
in lignifying tissues in Arabidopsis.
• Laccase activities were detected during xylem lignification.
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23. PEROXIDASE- CATALYZED LIGNIN
POLYMER FORMATION
• Peroxidases have been shown to catalyse the formation of
DHPs efficiently in vitro using H2O2 and monolignols .
• The enzymatic activity of peroxidases to produce DHPs
appears to be more specific to coniferyl Alcohol.
• Like Laccases, peroxidases are expressed in lignifying
vascular tissue.
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24. SECOND SUBSTRATE REQUIREMENT
– H2O2 OR O2
• Besides the lignin monomer, peroxidases and laccases
require additional substrates – hydrogen peroxide (H2O2) and
molecular oxygen (O2) respectively – to form monolignol
radicals.
• During anoxia/hypoxia, the triggered arrest of plant growth is
coupled to an increase of H2O2, as well as an increase of
lignin in both the Casparian strip and the sclerenchyma cells .
• The production of apoplastic H2O2 derives from a two-step
enzymatic process using nicotinamide adenine dinucleotide
phosphate hydrogen oxidase (NADPH oxidase) and
superoxide dismutase . 10/11/2018Department of Plant Biotechnology 24

25. HIGH ISOELECTRIC POINT
SUPEROXIDE DISMUTASE (SOD)
• Superoxide dismutases , divided into three different forms
(CuZn-SOD, Fe-SOD and Mn-SOD), catalyse the dismutation
of toxic superoxide radicals produced by NADPH oxidase into
O2 and H2O2 substrates which can be used by laccases or
peroxidases.
• Showed distinct subcellular localization in the secondary cell
walls of xylem TEs and fibres, as well as in the middle lamella
of surrounding xylem parenchyma cells.
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26. GENERAL LIGNIN POLYMERIZATION.
LIGNIN MONOMERS ARE EXPORTED ACROSS THE PLASMA
MEMBRANE BY PASSIVE DIFFUSION , EXOCYTOSIS (VESICLE) OR
THROUGH ABC TRANSPORTERS .
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27. LIGNIN POLYMERIZATION
LACCASE AND PEROXIDASE ..?
• In Arabidopsis thaliana, the Casparian strip lignification
depends on peroxidases , whereas xylem TE and fibre
lignification are mostly dependent on laccases.
• Laccases and peroxidases do not function redundantly .
• The peroxidases form rigid cross-links between lignin,
hemicelluloses and extensins in the secondary cell wall , once
the initial lignin polymers/oligomers are made by laccases.
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28. LIGNIN FORMATION DURING THE
DIFFERENTIATION OF SPECIFIC CELLS
• Lignification is an integral part of the differentiation process of
several specialized cell types.
• Lignin deposition exhibits differences in :-
1. Its timing during the cell differentiation process .
2. Its dependency on specific enzymes and substrates.
3. Its sub-cellular deposition in the cell wall.
4. Its monomeric composition .
5. Its autonomy for the production of the required
enzymessubstrates.
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29. 1. LIGNIFICATION OF XYLEM
TRACHEARY ELEMENTS
• The vascular and mechanical support functions of TEs is established
by the removal of the cell cytoplasm by programmed cell death and
the reinforcement of the cell side walls with a lignified secondary cell
wall.
TE differentiation from the elongated cells of the meristematic
cambium progresses sequentially with :-
1. A microtubule-guided cellulose and xylan secondary cell wall
deposition.
2. The cell autonomous programmed cell death.
3. The post-mortem lignification of the TE secondary cell wall and the
autolysis of the cell content.
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30. • This post-mortem lignification of TEs operates by the co-
operative supply of lignin monomers and ROS by both the
surrounding precursor cells and xylem parenchyma.
• Post-mortem lignification of TEs have evolved to withstand the
increasing pressure in the stem by prolonged lignification of
the TEs along with the increase in height and girth of the
stem.
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32. 2. LIGNIFICATION OF XYLEM FIBRES
• Sclerenchyma fibres are believed to derive from a primitive
ancestral xylem cell which evolved into TEs mainly for the sap
conduction and fibres for mechanical support.
• In contrast to TEs, fibres do not require cell death to achieve
their structural support function.
• Lignin oxidative polymerization in sclerenchyma fibres
depends on multiple laccases as well as peroxidases .
(Shigeto et
al.,2013).
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34. 3. LIGNIFICATION OF ROOT
ENDODERMAL CELLS
• Endodermal cells constitute a tightly bound root cell layer, regulating the flux of
compounds from the root cortex in contact with the rhizosphere to the root
medullar vascular system.
• The differentiation of these cells derives from the root tip and, once cells have
fully elongated, then the Casparian strip is formed by:
Establishing specific polarized domains, named CASPs, which will delimit the
sites of the Casparian strips both at the plasma membrane & in neighboring
apoplastic space.
NADPH oxidase is targeted to the CASP domain and is exported into the
CASP- delimited apoplastic space.
 Lignin monomers are secreted in the apoplast, some by an unpolarized ABC
transporter.
Lignin is polymerized in the apoplastic space by the CASP domain using
peroxidases, Apo plastic monomers and H2O2.
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36. DIFFERENT LEVELS OF CO-
OPERATIVE AND
AUTONOMOUS
LIGNIFICATION DEPENDING
ON THE CELL TYPES.
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Biotechnology
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37. CONCLUSIONS
• Lignin is associated with many different specialized cells to fulfill
specific physiological functions, but exhibits distinct properties for
each cell type.
• Depending on the desired lignin properties for the cell function,
specific substrates and enzymes will be produced in distinct cell
types, although a high plasticity for both substrates and enzymes
will still be retained.
• Depending on the different cell types, lignification will progress
autonomously and co-operate with the surrounding cells to
ensure full lignification and to adapt to environmental changes.
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38. REFERENCES
• Jaime Barrosy, Henrik Serk1, Irene Granlundz and Edouard
Pesquet Annals of Botany 115: 1053–1074, 2015.
• Weng JK, Chapple C. 2010. The origin and evolution of lignin
biosynthesis. New Phytologist 187: 273–285.
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Thank you..