Nitrogen Fixing Cereals – How close are

we?

Vidushi Rastogi
Roll No. 5014
 Nitrogen
One of the macronutrients.

Fixed N2 - Limiting factor for the plant growth.

Expected requirement of N-fertilizer by 2015 A.D. for 3 major

crop grasses will be 43.3 mill t.(FAO,U.N.)

Production & application of N-fertilizer

-major source of pollution.
-major consumption of energy.

“Need to augment cereal production- humans hit 9 billion by 2050

A.D.” (U.N. Population Division)
 What is Nitrogen Fixation?
Reduction of Dinitrogen to Ammonia
N2 is an inert gas- Ammonia is usable form

N2
in atm.

Chem. Free living flora

Fixation
Associative flora
Symbiotic flora

Bio. Phy.
Fixation Fixation
 N-Fixing Cereals - How it started?
Cereals were known to form AM symbiotic relationship with fungi.

-e.g. Rice forms AMS with Glomus intraradices , Gigaspora rosea ,

Piriformospora indica.

It was found that – The Symbiotic Pathway is common for the

formation of both RLS & AMS.

Since this signalling pathway is present in cereals , it should be

feasible to engineer this signalling pathway for cereal recognition
of nitrogen fixing bacteria.
(Gleason ,2006;Tirichine ,2006)
RLS- a relationship between diazotrophic microflora &
angiosperms

“Symbiosis between legumes & Rhizobia evolved approx. 60-70

mill y ago.”

Flavonoids
Lipochito-
oligosaccharide

-Kistner & Parniske , 2002

Activation Cortical
of gene cell
expression Calcium division
Spiking

-Oldroyd and Downie , 2008

 RLS in cereals- Is it doable?

There are evidences that :

Much of the genetic information required for BACTERIAL
COLONISATION & NODULE ORGANOGENESIS are present in
non-legumes.

-Messinese et al,2007
 Two avenues of RLS

Root Hair Dependent

- Advance & prominent
- Bacterial entry via root hair cells
Root Hair Independent
- Primitive.
- Bacterial entry at points
-of epidermal damage where lateral roots emerge
-between epidermal cells at base of root hairs
-Involves less of the sym genes products

-Sprent ,2007 ;Karas et al 2005

 RLS – The Mechanism
Infection pocket

Infection Thread
 RLS- The molecular dialogue

Study on model legumes

Lotus japonicus (with Mesorhizobium loti)

Medicago truncatula(with Mesorhizobium truncatula)

Nod factors are perceived by LysM-RLKs. C D
-LysM-RLKs bind NAG
A B
LysM-RLKs are present within NFRs, the heterodimeric
complexes.
-NFR1(L. japonicus)
-NFR5(M.truncatula)

8 genes act downstream of Nod factor receptors .

-SYMRK
-NUP85,NUP133,NENA
-CASTOR & POLLUX
-CCaMK
-CYCLOPS

Signal is transduced downstream of CCaMK via

nodulation specific transcription factors.
-NSP1,NSP2,ERN
 RLS- Current understanding

NOD

SymRK CCaMK
NFR

P P
P Ca

Ca

Nucleus

Stougaard et al,2010
Restoration of symbiotic defects in Lotus japonicus
mutants by Rice orthologs
-Banba et al,Plant Cell Physiology 2008

Three rice CSP gene orthologs were identified.

- OsCASTOR

- OsPOLLUX

- OsCCaMK.
 Structural comparisons of CASTOR, POLLUX and CCaMK

Numerals indicate local a.a. sequence identity (%)

A & B. TM=Transmembrane Helix , RCK=Regulation of Conductance in K

channels

C. PK= Protein Kinase , CAM=Calmodulin binding domain, EF=EF hand , Thr-

site of phosphorylation.
Transgenic roots were selected by using GFP fluorescence as
marker.

Presence of endosymbiotic bacteria was demonstated by

red fluorescence ( Ds-Red ) .
 Ljccamk + LjCCaMK
(Nodulation phenotypes)

RLS restored in Ljccamk mutants

Mature Nodule formed on Ljccamk roots
transformed with LjCCaMK

Pink color Red fluorescence

(Leghaemoglobin) in nodule centre
 Ljccamk + OsCCaMK
(Nodulation Phenotypes)

RLS restored in Ljccamk mutants

Mature Nodule formed on Ljccamk roots
transformed with OsCCaMK

Pink color Red fluorescence

(Leghaemoglobin) in nodule centre
 Similar studies were done with CASTOR & POLLUX.

The experiments showed that :

- OsCASTOR could fully restore rhizobial symbiosis in LjCASTOR

mutant of Lotus.

- But OsPOLLUX is not fully compatible with LjPOLLUX

Hence function of only CCaMK & CASTOR is conserved

between rice & Lotus.
- Though structures of all three are conserved between rice &
Lotus.
 Tomato & Rice SYMRK cannot or partially
complement bacterial endosymbiosis formation
-Markmann,Giczey,Parniske(PLoS 2008)

SYMRK is involved in generation of Ca signals through

autophosphorylation.

There is an exceptional diversification in genes among

different species.

Predicted extracellular portion of SYMRK occurs in 3 versions

of domain composition .
 Structure of SYMRK

Element L . japonicus O .sativa

Exons 15 12

LRR motifs 3 2

NEC domain Extended Short

Amino acids 923 576

 L .japonicus line symrk-10 was used.

symrk-10 carries a mutant allele encoding a kinase dead SYMRK

version.

Lotus symrk-10 roots were transformed with

-control vector(lacking a SYMRK expression cassette)
-LeSYMRK
-OsSYMRK

Root systems were inoculated with Mesorhizobium loti expressing

DsRed for 4 weeks.
Symrk-10+control vector

No nodules
Symrk-10 + LeSYMRK

No nodules
 Symrk-10 + OsSYMRK

Nodules not fully developed but nodule primordia

present
 Symrk-10 + OsSYMRK

Nodule primordia non colonised by bacteria Small nodules colonised with

bacteria
Bacteria proliferating on primordial surface. But no pinkish coloration
(rare case)
The SYMRK function is not completely conserved between Lotus
& Rice.

Os CSP orthologs donot completely compensate for RLS,

suggesting molecular evolution occurred in CSP genes to
acquire RLS.

SYMRK is believed to be Epistatic to other genes.

SYMRK version of non-legumes does not suffice for RLS because

of presumed :
-amino acid sequence polymorphism.
-lack of exons required for bacterial endosymbiosis.
 Common SYM genes in Rice & A. thaliana
Legume Rice sym Phenotype of legume root A .thaliana sym
sym symbiosis mutant transformed
with Rice orthologue
SymRK OsSymRK Nod(-) …
NUP85 OsNUP85 n.d. AtNUP85
NUP133 OsNUP133 n.d. AtNUP133
NENA OsNENA n.d. AtNENA
CASTOR OsCASTOR Nod(+) …
POLLUX OsPOLLUX Type1,Inf(-) AtPOLLUX
CCaMK OsCCaMK Nod(+) …
CYCLOPS OsCYCLOPS Nod(+) …
VAPYRIN OsVAPYRIN n.d. …

(+) restored Nod=nodulation TypeI =small bump

(-) not restored Inf =bacterial infection n.d. = not determined
 Rays of hope
 Roots of certain Rice cultivars exude compounds which induce
, albeit to a low extent transcription of nod genes of
Rhizobium sp. NGR234. (Reddy et al,1998)

 In Rice Nod factors can induce the expression of a reporter

gene under the control of M.truncatula ENOD12 promoter.
(Reddy et al,1998)

 Photosynthetic Bradyrhizobia are natural endophytes of the

African wild rice Oryza breviligulata. (Clemence et al, 2000)

 Application of Nod factors enhanced germination & early

growth of Rice. (Prithviraj et al, 2003)
 What our Rice can do ?
Let the Rhizobia colonise its rhizosphere.

Reactivate cortical cells in the presence of a microsymbiont .

Accept penetration of the symbiont in host cells .

Synthesize membranous interface with the symbiotic partner

to facilitate metabolic exchange .
 Path ahead

Predicting the implications of rhizobial recognition in cereals-

 will this lead to developmental changes associated with

nodulation?
 will it allow colonisation by rhizobial bacteria?
 will there be no response implying further engineering
processes are necessary?

Engineering N-fixing cereals should involve a gradual

enhancement of rhizobial colonisation of root & stepwise
improvement in Nitrogen Fixing Efficiency.
 Skepticism
Rather than putting resources into N-fixation , it would be more productive to
improve Nutrient Uptake Efficiency of cereals.

Much of the plant’s N-metabolism & transport system would also have to be
engineered.

It will take a bunch of successful engineering transformations to get a so called

N-fixing cereal.

“If I could put all the genes needed to create a N-fixing plant into
corn. I would probably end up with a plant resembling soya”
-Ernie Jaworski
 Enthusiasm
We will not need to transfer to cereals every genetic component defined in
legumes for nodulation.

We just need to simulate what has happened in nature 70 mya .

Cereals require much less N than legumes,so we need not pressurize

engineered symbiosis for high rate of fixation.

It would be the most significant contribution of Biotechnology to agriculture.

“ Nature herself had brought them in a relationship, we

just need to make it more intimate.”
-Edward Cocking
 Conclusion
Engineering cereals to make them able for rhizobial symbiosis
is a feat requiring courage, resources & time.

The anticipated results are overly exciting that it’s a work

worth it.

Realizing this academic exercise into a field competent

innovation may turn the agriculture upside down, for a good.