Plant Cell Wall Polymers - American Chemical Society

Chapter 11

Biogenesis and Structure of Macromolecular Lignin in the Cell Wall of Tree Xylem as Studied by Microautoradiography

Downloaded by PURDUE UNIV on November 29, 2016 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch011

Noritsugu Terashima and K. Fukushima Faculty of Agriculture, Nagoya University, Nagoya 464—01, Japan

Specific labeling of the protolignin of various trees was achieved by administration of appropriate H-labeled monolignol glucosides to differentiating tree xylem. The process of deposition of each labeled precursor in the developing cell wall was visualized by means of high resolution microautoradiography. It was found that the monolignol (and polysaccharide which affects polymerization) changed with both type and age of the individual cell. The incorporation of monolignols into the protolignin macromolecule occurred in the order of increasing complexity, i.e., p-hydroxyphenyl-, guaiacyl- and syringylpropane units were deposited successively. It was noted that the lignin deposited in the early stages of cell wall development in the compound middle lamella region contained more condensed structures than that formed in the later stages in secondary wall. It was concluded that lignin formation occurs under definite biochemical regulation to give a macromolecule which is heterogeneous in structure and specific in its morphological location. 3

It is w e l l k n o w n t h a t the s t r u c t u r e , d i s t r i b u t i o n a n d properties o f p r o t o l i g n i n i n cell walls vary a c c o r d i n g t o cell t y p e a n d m o r p h o l o g i c a l l o c a t i o n . T h i s is based u p o n extensive studies o n t o p o c h e m i c a l properties o f l i g n i n u s i n g various methods such as u l t r a v i o l e t m i c r o s c o p i c p h o t o m e t r y (1,2), b r o m i n a t i o n - S E M - E D X A (3) a n d other p h y s i c a l or c h e m i c a l analyses o f isolated tissue fractions (4). In w o o d y g y m n o s p e r m s , there are significant differences i n the d i s t r i b u t i o n , r e a c t i v i t y a n d p h y s i c a l properties o f p r o t o l i g n i n s f o u n d i n the c o m p o u n d m i d d l e l a m e l l a a n d the secondary w a l l (1-3). A d d i t i o n a l l y , v a r i a t i o n s between l i g n i n s i n vessels a n d fibers have also been noted (3). A l l o f these 0097-6156/89/0399-0160$06.00/0 © 1989 American Chemical Society

Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

11.

161

Mocromoleculor Lignin

TERASHIMA & FUKUSHIMA


observations are p o t e n t i a l l y e x p l i c a b l e b y factors such as (i) differences i n m o n o m e r i c c o m p o s i t i o n , (ii) differences i n i n t e r u n i t linkages between i n d i v i d u a l l i g n i n m o n o m e r s , (iii) linkages between l i g n i n a n d c a r b o h y d r a t e s , a n d (iv) v a r i a t i o n s i n d i s t r i b u t i o n (frequency a n d l o c a l i z a t i o n ) of s t r u c t u r a l moieties i n the l i g n i n m a c r o m o l e c u l e . M a n y approaches have been t r i e d to solve these p r o b l e m s , p a r t i c u l a r l y d e g r a d a t i o n analyses such as a c i d o l y s i s a n d o x i d a t i o n . However, these have afforded o n l y l i m i t e d i n f o r m a t i o n o n the t o p o c h e m i c a l n a t u r e of l i g n i n . H e n c e , more d e t a i l e d i n f o r m a t i o n a b o u t the m a c r o m o l e c u l a r s t r u c t u r e o f p r o t o l i g n i n can o n l y be o b t a i n e d b y s u i t able n o n - d e g r a d a t i v e analyses. In t h i s c h a p t e r , the results o b t a i n e d f r o m the c o m b i n e d use of spe cific r a d i o l a b e l i n g o f l i g n i n i n p l a n t tissue a n d m i c r o a u t o r a d i o g r a p h y are discussed. Materials and

Methods

Radioactive Precursors for Microautoradiography. Several l i g n i n precursors were labeled w i t h H or C at specific p o i n t s . T h e s e precursors i n c l u d e d p - c o u m a r i c , f e r u l i c , a n d s i n a p i c acids, a n d the /?-D-glucosides o f the three m o n o l i g n o l s , p - c o u m a r y l , c o n i f e r y l , a n d s i n a p y l alcohols. H - l a b e l e d pre cursors were u s u a l l y prepared b y replacement of the h y d r o g e n at p o s i t i o n 2 of the a r o m a t i c r i n g w i t h H , since essentially a l l ( > 98%) of the H at t h i s p o s i t i o n is r e t a i n e d d u r i n g l i g n i n f o r m a t i o n (5). P r e c u r s o r s labeled at C - 5 or C - 6 of the a r o m a t i c rings can also be e m p l o y e d for d e t e r m i n a t i o n of the extent of s u b s t i t u t i o n reactions at these p o s i t i o n s ( T e r a s h i m a , t h i s v o l u m e ) . A f t e r i n c o r p o r a t i o n o f precursors i n t o g r o w i n g p l a n t s , m i c r o a u t o r a d i o g r a m s of r a d i o l a b e l e d tissue sections were p r e p a r e d , a n d these gave s e m i q u a n t i t a tive i n f o r m a t i o n r e g a r d i n g t h e i r d e p o s i t i o n w i t h i n the cell w a l l . T h i s was o b t a i n e d b y d e t e r m i n a t i o n of their a c t i v i t i e s u s i n g the technique of silver g r a i n c o u n t i n g (6). G e n e r a l l y , the H - l a b e l e d precursors are m o r e s u i t able for such s e m i q u a n t i t a t i v e e s t i m a t i o n s t h a n their C - 1 4 c o u n t e r p a r t s , because low energy ^ - e m i t t e r H can afford a h i g h r e s o l u t i o n a u t o r a d i o g r a m . T h e specific r a d i o a c t i v i t y o f the precursors m u s t , however, be h i g h enough ( > ΙμΟι/μπιοΙ) to o b t a i n a g o o d m i c r o a u t o r a d i o g r a m i n a reason able p e r i o d of t i m e ( < 3 weeks). O n the other h a n d , precursors labeled with C at p o s i t i o n s i n the a r o m a t i c r i n g are more desirable for t r a c i n g the fate of p a r t i c u l a r s t r u c t u r a l u n i t s d u r i n g various reactions. P r e c u r s o r s labeled at s i d e - c h a i n carbons w i t h C - 1 4 c o u l d also be used since s i d e - c h a i n cleavage reactions f r o m the l i g n i n m a c r o m o l e c u l e r a r e l y o c c u r d u r i n g l i g n i n f o r m a t i o n (5). 3

1 4

3

3

3

3

3

1 4

Administration of Precursor. P r e c u r s o r s were a d m i n i s t e r e d to p l a n t s ac c o r d i n g to p r e v i o u s l y described procedures (6). Shoots of 2-3 year o l d trees were o b t a i n e d d u r i n g J u n e or J u l y , w h e n the rate o f t h i c k e n i n g g r o w t h was highest. A V - s h a p e d groove, 2 m m wide a n d 5 m m l o n g , was m a d e w i t h a razor blade i n a c i r c u m f e r e n t i a l d i r e c t i o n o n the shoot, so t h a t the b o t t o m of the groove reached the d i f f e r e n t i a t i n g x y l e m . F i n e glass w o o l was packed i n t o the groove, a n d a s o l u t i o n of the precursor (8-10 μΟί, 1 m g i n 100


162

PLANT C E L L W A L L POLYMERS


μΐ phosphate buffer, p H 7.0) was added dropwise to the fine glass w o o l , a n d allowed to metabolize for 3 h . A d r o p of 3 % g l u t a r a l d e h y d e i n phos phate buffer ( p H 7.0) was then added to the groove, a n d a s m a l l block of x y l e m tissue near the groove was cut a n d fixed again i n 3 % g l u t a r a l d e h y d e overnight i n the refrigerator. P a r t of the x y l e m tissue was d e h y d r a t e d b y means of a graded e t h a n o l series a n d e m b e d d e d i n e p o x y resin prepared b y m i x i n g Q u e t o l 812 (100 g, N i s s i n E M C o . L t d . , T o k y o ) , m e t h y l n a d i c a n h y d r i d e (89 g) a n d 2 , 4 , 6 - t r i - [ d i m e t h y l a m i n o m e t h y l ] - p h e n o l (1.7 g). Preparation of Microautoradiogram. M i c r o a u t o r a d i o g r a m s were prepared a c c o r d i n g to the procedure described earlier (10). T w o μπι t h i c k transverse sections were cut f r o m the embedded x y l e m tissue u s i n g a R e i c h e r t - J u n g S u p e r c u t 2050 m i c r o t o m e e q u i p p e d w i t h a glass knife. These were t h e n m o u n t e d o n glass slides a n d covered w i t h K o d a k A R - 1 0 s t r i p p i n g film. T h e glass slides were stored i n a refrigerator f r o m 3 weeks to 1 year as r e q u i r e d , f o l l o w i n g w h i c h they were developed w i t h K o d a k D - 1 9 a n d fixed w i t h F u j i F i x . T h e sections were s t a i n e d w i t h t o l u i d i n e blue O , a n d p h o t o m i c r o g r a p h s were m a d e u s i n g a Zeiss I B A S 1 image a n a l y z e r . A p o l a r i z a t i o n microscope, O l y m p u s P O S , was used to observe the d e p o s i t i o n of cellulose m i c r o f i b r i l s d u r i n g secondary w a l l f o r m a t i o n . Results and Discussion Selectivity of Labeling. T h e degree of selectivity of l a b e l i n g of a specific l i g n i n m o n o m e r was e s t i m a t e d f r o m the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o the a r o m a t i c aldehydes, o b t a i n e d by nitrobenzene o x i d a t i o n of labeled w o o d tissue. A m o n g m a n y c o m p o u n d s tested, labeled ferulic a c i d a n d coniferin were s u i t a b l e precursors for l a b e l i n g of pine l i g n i n (5,7). Indeed, it was recently s h o w n t h a t exogeneously a p p l i e d ferulic a c i d was u t i l i z e d b y c u c u m b e r seedlings as a n effective precursor for endogeneous l i g n i f i c a t i o n (8). A d d i t i o n a l l y , under special feeding c o n d i t i o n s , labeled s i n a p i c a c i d can be e m p l o y e d to p r e d o m i n a n t l y l a b e l the s y r i n g y l moiety of p o p l a r l i g n i n (9). However, highest selectivity i n l a b e l i n g is n o r m a l l y achieved b y a d m i n i s t r a t i o n of p - g l u c o c o u m a r y l a l c o h o l , coniferin or s y r i n g i n (6,10). Biogenesis of Lignin in Woody Angiosperms. W h i l e the l i g n i n precursors, coniferin a n d s y r i n g i n have been found i n the c a m b i a l sap of a few a n giosperms belonging to the M a g n o l i a c e a e a n d Oleaceae families (11), the glucoside of p - c o u m a r y l alcohol has never been detected. Nevertheless, these three glucosides were prepared i n r a d i o l a b e l e d f o r m as c a n d i d a t e m o n o l i g n o l precursors. T h u s , when coniferin a n d s y r i n g i n , labeled w i t h H at p o s i t i o n 2 of the a r o m a t i c r i n g , were a d m i n i s t e r e d to Magnolia kobus D C , each was efficiently i n c o r p o r a t e d into its l i g n i n d u r i n g a l l phases of l i g n i f i c a t i o n (10). O n the other h a n d , the i n c o r p o r a t i o n of p - g l u c o c o u m a r y l a l c o h o l was o n l y observed d u r i n g early stages of cell w a l l f o r m a t i o n (12). 3

A d d i t i o n a l experiments revealed the f o l l o w i n g : w h e n a g r o w i n g s t e m of lilac (Syringa vulgaris L . ) was a d m i n i s t e r e d the same H - l a b e l e d m o n o l i g n o l glucosides, each was efficiently i n c o r p o r a t e d into the n e w l y f o r m e d 3


11.

TERASHIMA & FUKUSHIMA

Macromokculor

Lignin

163


x y l e m , a l t h o u g h at different stages o f cell w a l l f o r m a t i o n as revealed b y m i c r o a u t o r a d i o g r a p h y ( F i g . 1). A g a i n , p - c o u m a r y l a l c o h o l was i n c o r p o r a t e d o n l y d u r i n g the earliest stages o f cell w a l l development. F i g u r e 2 shows a n enlarged p h o t o g r a p h of a p a r t of t h i s a u t o r a d i o g r a m . A s c a n be seen, the d i s t r i b u t i o n of silver g r a i n s i n d i c a t e s t h a t the p - h y d r o x y p h e n y l l i g n i n c o m ponents were deposited m a i n l y i n the m i d d l e l a m e l l a regions, a n d were not present i n the secondary w a l l . O n the other h a n d , g u a i a c y l l i g n i n f o r m e d c o n t i n u o u s l y f r o m the early to the later stages, a n d i n b o t h the vessels a n d fiber w a l l s . Interestingly, s y r i n g y l l i g n i n was deposited m o s t l y i n the seco n d a r y w a l l o f fibers, a l t h o u g h a s m a l l a m o u n t was also used for f o r m a t i o n of m i d d l e l a m e l l a l i g n i n ( F i g s . 1 a n d 3). S i m i l a r trends were observed i n m a g n o l i a (10) a n d p o p l a r (9). Biogenesis of Lignin in Woody Gymnosperms. F i g u r e 4 shows the d i s t r i b u t i o n o f silver g r a i n s i n the n e w l y f o r m e d x y l e m of the g y m n o s p e r m , J a p a n e s e b l a c k p i n e (Pinus thunbergii P a r i . ) a d m i n i s t e r e d the three H - l a b e l e d m o n o l i g n o l glucosides as before, a n d U D P - g l u c u r o n i c a c i d - [ g l u c u r o n y l - U C ] a n d a n d G D P - m a n n o s e - [ m a n n o s e - l - H ] . T h e s e nucleotides are c o n s i d ered to be precursors of p e c t i n a n d hemicellulose (13). F i n a l l y , w h e n the labeled U D P - g l u c u r o n i c a c i d was a d m i n i s t e r e d , a large p a r t of the silver grains was l o c a l i z e d m a i n l y o n the cell walls f o r m e d d u r i n g the earliest stage of x y l e m differentiation a n d p a r t l y o n those f o r m e d d u r i n g the next stage. T h i s c a n be a s c r i b e d to the i n i t i a l d e p o s i t i o n of p e c t i c substances a n d hemicelluloses derived f r o m U D P - g l u c u r o n i c a c i d . O n the other h a n d , the a c t i v i t y f r o m G D P - m a n n o s e was i n c o r p o r a t e d l a r g e l y d u r i n g secondary w a l l f o r m a t i o n . A s p r e v i o u s l y discussed, the l i g n i n f o r m e d d u r i n g the early stages of cell w a l l development i n the m i d d l e l a m e l l a a n d cell corner region contains more condensed u n i t s t h a n t h a t f o r m e d d u r i n g later stages i n the secondary w a l l ( T e r a s h i m a , t h i s v o l u m e , a n d ref. 6). T h i s finding c a n n o w be r a t i o n a l i z e d b y the fact t h a t w h e n c o n i f e r y l a l c o h o l was p o l y m e r i z e d i n p e c t i n a n d m a n n a n gel in vitro, the " L C C " f r a c t i o n f o r m e d i n p e c t i n contains more condensed u n i t s t h a n t h a t f o r m e d i n m a n n a n ( T e r a s h i m a , t h i s v o l u m e , a n d ref. 14); i.e., the t y p e o f p o l y s a c c h a r i d e influences p r o d u c t f o r m a t i o n . A l l three m o n o l i g n o l s were i n c o r p o r a t e d i n t o l i g n i n at different stages o f cell w a l l f o r m a t i o n . A u t o r a d i o g r a m s revealed t h a t 62, 38 a n d 2 4 % of the silver g r a i n s , assigned to p - h y d r o x y p h e n y l , g u a i a c y l a n d s y r i n g y l u n i t s respectively, were i n the c o m p o u n d m i d d l e l a m e l l a ( F i g . 4). T h i s is i n g o o d agreement w i t h the o b s e r v a t i o n m a d e o n b l a c k spruce w o o d t h a t m i d d l e l a m e l l a l i g n i n contains a r e d u c e d m e t h o x y l content w h e n c o m p a r e d w i t h secondary w a l l l i g n i n (15). S y r i n g a l d e h y d e has been r e p o r t e d t o be one of the m i n o r components of the aldehyde m i x t u r e o b t a i n e d b y n i t r o b e n zene o x i d a t i o n of l i g n i n i n v a r i o u s conifers (16). A s m a l l s y r i n g y l content has also been detected d u r i n g the t h i o a c i d o l y s i s of pine (Pinus pinaster) compression w o o d (17). I n a related s t u d y , w h e n Japanese b l a c k pine (P. thungergii) w o o d was subjected to nitrobenzene o x i d a t i o n , s y r i n g a l d e h y d e was o b t a i n e d i n a considerable a m o u n t , a n d represented a b o u t 5 m o l % of the t o t a l aldehyde m i x t u r e (18). Interestingly, i n the cell w a l l of t h i s 3

1 4

3



3

3

3

F i g u r e 1. M i c r o a u t o r a d i o g r a m s of differentiating x y l e m of Synnga vulgaris L . a d m i n i s t e r e d w i t h [arom. r i n g - 2 - H ] p - g l u c o c o u m a r y l a l c o h o l (a); [arom. r i n g - 2 - H ] coniferin (b); a n d [arom. r i n g - 2 - H ] s y r i n g i n (c).



3

Figure 2. A part of microautoradiogram of differentiating xylem of Syringa vulgaris L . administered [arom. ring-2- H] p-glucocoumaryl alcohol.

3

Figure 3. A part of microautoradiogram differentiating xylem of Syringa vulgaris administered [arom. ring-2- H] syringin.


166

PLANT C E L L W A L L

POLYMERS

Relative density of silver grai ns Precursor: « syringin-Ca.r.-2- HI J

• hw"

^

•

•

coniferin-ta.r.-2- H] J

p - g l u c o c o u m a r y l alcohol-Ca.r-2 ^Start of S 3 ^ o r m a t i o a ' ' ^ GDP-mannose-Cmannose-1- H] 3


^1 Tormatjon' ,'' ' / U D P - g l u c u r o n i c acid-Cglucuronyl-U-'^C] 100 200 400 600 Distance f r o m c a m b i u m

800 jjm

F i g u r e 4. D i s t r i b u t i o n of silver grains i n m i c r o a u t o r a d i o g r a m s of different i a t i n g x y l e m of pine a d m i n i s t e r e d w i t h precursors of l i g n i n a n d h e m i c e l l u lose. p i n e , the s y r i n g y l moieties are d i s t r i b u t e d m a i n l y i n the i n n e r layer of the secondary w a l l , even t h o u g h its content is very low. Concluding

Remarks

T h e results o b t a i n e d b y a u t o r a d i o g r a p h y a n d other studies (6,7,10, a n d T e r a s h i m a , t h i s v o l u m e ) , i n d i c a t e d t h a t the s t r u c t u r e of the p r o t o l i g n i n m a c r o m o l e c u l e is heterogeneous w i t h respect to m o n o m e r c o m p o s i t i o n , d i s t r i b u t i o n of i n t e r u n i t linkages (to f o r m condensed s u b s t r u c t u r e s ) , a n d association w i t h carbohydrates. However, p r o t o l i g n i n is not a disordered c o p o l y m e r of various m o n o l i g n o l s . Instead, m a c r o m o l e c u l a r f o r m a t i o n occurs i n a b i o c h e m i c a l l y r e g u l a t e d m a n n e r , a n d the heterogeneous n a t u r e of p r o t o l i g n i n is a n a t u r a l a n d i n e v i t a b l e consequence of its u n i q u e m e c h a n i s m of biogenesis. F i g u r e 5 s u m m a r i z e s the successive d e p o s i t i o n of cell w a l l c o m p o n e n t s a n d their irreversible assembly to f o r m a l i g n i f i e d cell w a l l i n tree x y l e m . T h e causes of p r o t o l i g n i n heterogeneity c a n be e x p l a i n e d as follows. 1. T h e process of l i g n i f i c a t i o n is f u n d a m e n t a l l y c o n t r o l l e d by each i n d i v i d u a l cell. 2. L i g n i f i c a t i o n is preceded always by d e p o s i t i o n of cell w a l l p o l y s a c c h a rides, a n d m o n o l i g n o l p o l y m e r i z a t i o n occurs w i t h i n the c a r b o h y d r a t e gel, r e s u l t i n g i n the s t r u c t u r e of the p o l y l i g n o l . H o w e v e r , the t y p e of c a r b o h y d r a t e changes w i t h cell w a l l development stages, i.e., f o r m a t i o n of cell w a l l layers, as s h o w n i n F i g u r e 5. A s discussed, the p o l y m e r i z a t i o n of m o n o l i g n o l s i n p e c t i c substances i n e a r l y stages m a y be one of the reasons w h y the m i d d l e l a m e l l a a n d cell corner l i g n i n c o n t a i n s m o r e condensed s u b s t r u c t u r e s t h a n secondary w a l l l i g n i n . 3. T h e m o n o l i g n o l u t i l i z e d varies w i t h t y p e a n d age of the cell. Indeed, i n c o r p o r a t i o n of m o n o l i g n o l s into p r o t o l i g n i n occurs i n order of i n -



layers

Fiber

S 2

— S 3

—

•SW

-SW •CC -CML

•sw

G-

H

-*-Pectic substances Arabinogalactan Xylan • Mannan •Cellulose • Lignin iAgeing -p-Coumaryl alcohol Coniferyl alcohol-Sinapyl alcoholcc low degree of CML condensation

—

-^Mature cell wall

v

Figure 5. A schematic representation of the process of deposition of cell wall components and the heterogeneous formation of protolignin macromolecule. ML, middle lamella; CC, cell corner; P, primary wall; CML, compound middle lamella; S S2, and S3, outer, middle, and inner layer of secondary wall; H, G, and S, p-hydroxy-, guaiacyl-, and syringylpropane units.

Plant Cell Wall Polymers - American Chemical Society

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