Plant and Soil, Special Volume 1971, 225-228 Ms.

S V W - I 9

EFFECTS OF WATER STRESS ON NITROGEN

FIXATION IN ROOT NODULES
b y J. I. S P R E N T
Dept. of Biological Sciences, University of Dundee, Dundee, Scotland, United Kingdom

SUMMARY

W a t e r stress is k n o w n to a f f e c t t h e f o r m a t i o n a n d l o n g e v i t y of l e g u m i n o u s
r o o t nodules. W o r k in D u n d e e h a s s h o w n t h a t i t also a f f e c t s r a t e s of n i t r o g e n
f i x a t i o n in nodules, l i c h e n s a n d b l u e - g r e e n algae.
I n nodules, m o d e r a t e w a t e r stress slows d o w n a c e t y l e n e r e d u c t i o n , n i t r o g e n
r e d u c t i o n a n d r e s p i r a t o r y a c t i v i t y . T h e s e e f f e c t s are r e v e r s i b l e . M o r e s e v e r e
stress leads t o c o m p l e t e c e s s a t i o n of n i t r o g e n f i x a t i o n , v e r y low r e s p i r a t o r y
r a t e s a n d s t r u c t u r a l c h a n g e s w i t h i n t h e nodule. N o r e v e r s a l of t h e s e e f f e c t s
is f o u n d a n d t h e n o d u l e s are p r o b a b l y s h e d b y t h e p l a n t .
I t is s u g g e s t e d t h a t w a t e r s u p p l y h a s a m a j o r e f f e c t o n t h e a m o u n t of
n i t r o g e n f i x e d in t h e field, p a r t i c u l a r l y in t h o s e l e g u m e s w h i c h h a v e n o d u l e s
n e a r t h e soil surface.

Little work has been done on the effects of water on nitrogen-

fixing systems. The formation and longevity of root nodules has
been shown to be affected b y the availability of water in the soil 2 7
In Dundee, the effects of water on the physiology of nitrogen
fixation are being studied. All systems examined so far (legume
root nodules, non-legume nodules, blue-green algae and lichens)
have been shown to be sensitive to water supply, either in excess,
deficiency, or both. This report deals with some of the effects on
legume nodules.
Most of the work so far has been on detached nodules. Because
of their ability to reduce both acetylene and nitrogen for some
hours after detachment, they form a very convenient experimental
system 5. The effects of water stress on detached soybean nodules
fall into two categories, described in more detail elsewhere 6.
(a) where the fresh weight of nodules falls below 80 per cent of its
maximum (i.e. fully turgid) value, reduction of both acetylene and
226 j . I . SPRENT

nitrogen ceases, the r e s p i r a t o r y rate falls to a v e r y low level and

gross s t r u c t u r a l changes occur, The effects are irreversible and such
nodules p r o b a b l y degenerate in the i n t a c t plant.
(b) between 80 and 100 per cent m a x i m u m fresh weight, a sub-
m a x i m a l rate of fixation is m a i n t a i n e d , related to the w a t e r content.
This ix also a c c o m p a n i e d b y a lowered r e s p i r a t o r y rate. The effects
are reversible when nodules are stressed whilst still a t t a c h e d to the
p a r e n t root system.
T y p i c a l results from intact plants are sh~wn in Table 1. Three
sets of plants were used, in v;trious states of water stress. Samples
of nodules were relnovcd and their ~lccl\,It~lte rt,duction r~ttes
measured. The rool systems W e l - e t l l e l l immersed in water for 45
rains, alltl fro-thor samples ~>f nodnh's tt.Mcd. The, h'ss severely
stressed nodules are st'ell to I)t' fully l t ' e o v t , r e d , tilt" mor~' severely
stressed oIles less so, p r o b a b l y becallse it prop~rtion of t h e m were
stressed b e y o n d the, recovery point.

T A LI 1.1,; I

A c e t y l e n e r e d u c i n g a c t i v i t y of nodule~ t a k e t l f r o m w a t e r - M r e s s e d p l m l t s before a n d
a f t e r t h e r o o t s w e r e i m m e r s e d in w a t e r f o r 45 m i n

Appearance Watering t'ere, e n t a ~ e % max* n M C2H4/

~f pl;mt~ water l:W mg DW/br
in n o d u l e s

S t r o n g l y wilted before 379 84.2 11.1

after 425 94.3 26.8

Slightly wilted before 387 86.0 60.8

after 446 99.0 115.9

Not wilted before 456 I01.0 114.4

after 445 99.0 127.9

* based on maximum w a t e r c o n t e n t of 450%

We h a v e also tested the effects of waterlogging on nitrogen

fixation. Nodules which are immersed in w a t e r reduce acetylene at
o n l y a v e r y low level 5, an effect entirely due to oxygen deficiency.
R e c e n t l y we h a v e found t h a t s o y b e a n nodules can survive for
several d a y s u n d e r water, whilst a t t a c h e d to the p a r e n t plant,
resuming their full rate of a c t i v i t y w h e n the w a t e r is drained off.
Tests with plants of the non-legume Myrica gale grown u n d e r
 W A T E R STRESS IN L E G U M E ROOT NODULES 227

natural waterlogged conditions have shown t h a t whilst under

water these nodules are also functioning at sub-maximal levels.
There are indications in this case t h a t nodules which have developed
under water are relatively more active when under water t h a n
those which have developed under better drained conditions.
It is thus clear that all aspects of water supply must be taken
into account when considering the total amounts of nitrogen fixed.
Temporary excesses or deficiencies can lead to greatly reduced
activity. Both effects can be related to oxygen uptake. In the
case of waterlogging, oxygen uptake is restricted purely as a result
of its diffusion rate being lower in water than in air. The effects
of water stress on oxygen uptake are likely to be of a more funda-
mental metabolic nature. Our observations on soybean, and photo-
graphs published in the literature on other species, indicate t h a t
amongst the actively nitrogen-fixing cells there is a network of
intercellular spaces and a mosaic of uninfected host ceils. The
intercellular space system probably serves to aerate the tissue 3
We have calculated that at least a quarter of the nodule volume
m a y be occupied by air space, ensuring that oxygen can reach the
central parts of a nodule. Even so, oxygen supply m a y limit the
rate of nitrogen fixation 1. B e r g e r s e n 1 suggested t h a t when
oxygen concentration is increased above atmospheric levels, the
initial effect is to stimulate host cell activity, resulting (presumably)
in increased supplies of reductant and/or ATP to the nitrogen
fixing bacteroids. Above a pO2 of 50 per cent, oxygen reaches the
bacteroids, which become aerobic and nitrogen fixation ceases.
These results suggest a close metabolic interaction between the
host and bacterial symbionts. Our observations indicate t h a t this
interaction is not only between the host cytoplasm and bacteroids
of the s a m e cell, but between the uninfected ceils and their infected
neighbours. Numerous plasmodesmata occur in the contiguous
walls and these are ruptured when nodules are water stressed
beyond recovery, i.e. when nitrogen fixation ceases. These un-
infected cells m a y well have a vital role to play in nodule metabo-
lism and possibly also in transporting material to the cortex for
subsequent export from the nodule. We are currently examining
less severely stressed nodules to see if there are a n y reversible
structural differences as found for maize root tips by other workers
in this field 4
228 W A T E R S T R E S S IN L E G U M E ROOT N O D U L E S

REFERENCES

1 B e r g e r s e n , F. J., J. Gen. Microbiol. 29, 113-125 (1962).

2 D o k u , E. V., Exptl. Agric. 6, 13-18 (1970).
3 G o o d c h i t d , D. J. and B e r g e r s e n , F. J., J. l~acteriol. 92, 204-213 (1966).
4 N i r , I. et al., Australian J. Biol. Sci. 23, 489-491 (1970).
5 S p r e n t , J. I., Planta (Berl.) 88, 372-375 (1969).
6 S p r e n t , J. I., New Phytol. 70, 9-17 (1971).
7 W i l s o n , J. K., J. Am. Soe. Agron. 23, 670-674 (1931).