Role of miRNA for Nutrient Use Efficiency in crops

ICAR-IndianAgriculturalResearchInstitute,NewDelhi
GP 691- Seminar
Exploration of miRNA Diversity for
Nutrient Use Efficiency in Crops
Seminar leader :
Dr . V S Hegde
Principal scientist
IARI , New Delhi
Speaker:
Rahul kumar
M.Sc first year
DIV. Of Genetics
IARI , New Delhi

Outline
 Introduction
 Need for improvement of Nutrient Use Efficiency
 Approaches for improving Nutrient use Efficiency
 Role of miRNA for Nutrient Homeostasis in Plants
 miRNA Diversity for Nutrient use Efficiency
 Case study
 Conclusion

Nutrient Use Efficiency (NUE) may be defined
as yield per unit input. In agriculture this is usually
related to the input of fertilizer, whereas in
scientific literature the NUE is often expressed as
fresh weight or product yield per content of
nutrient.
Nutrient Use Efficiency can be designated as
nutrient efficiency ratio, agronomic efficiency,
physiological efficiency, agrophysiological
efficiency, apparent recovery efficiency, and
utilization efficiency.
The nutrients most commonly limiting plant
growth are N, P, K and S. NUE depends on the
ability to efficiently take up the nutrient from the
soil, but also on transport, storage, mobilization,
usage within the plant, and even on the
environment.
NUTRIENT USE EFFICIENCY
Fig: The Uptake of nutrient by a plant

Paul et al,2015

Import of fertilizers of value Rs. 34600 crores (2012-2013)
Loss of 2900 Cr. In India due to losses of N Fertilizer
Human health issue- in Punjab and Haryana ground water samples had
Nitrate-N of >22mg/l.
1% increase in NUE in N and P will save Rs. 10056 million
Need To Enhance NUE
P. fixen , 2014

Agronomic Approaches
Physiological Approaches
Genetic Approaches
1.Conventional Approach
2.Genomic Approach
Approaches for improving NUE

 1st mi-RNA to be discovered in 1993 in
Lin-4 gene of C.elegans
 By Victor Ambros and Gary Ruvkun
 miRNAs that are20 -24nucleotides long
bind to specific complementary transcripts,
attenuating gene expression at the post-
transcriptional level or via translational
inhibition.
 miRNAs are vital for maintaining nutrient
homeostasis in plants by regulating the
expression of transporters that are involved
in nutrient uptake and mobilization.
ICAR-INDIANAGRICULTURALRESEARCHINSTITUTE
Role of miRNA in NUE
Victor Ambros and Gary Ruvkun

Fig: miRNA mediated signaling network for regulation of nutrient homeostasis
Paul et al,2015

Figure: Evolution of miRNA Diversity
Berezikov et al,2011

miRNA diversity related to NUE
Plant
tissue
Plant
species(r
eference)
Plant
tissue
Plant
species(r
eference)
156 SQUAMOSA
PROMOTER
BINDING PROTEIN-
LIKE (SPL)
transcription
factors
Shoot development .
Delayed vegetative phase
change
R (+) maize R (+) Arabidopsis Zhao et
al,2012
160 Auxin response
factors
Reduce auxin responsive
activities and the vegetative
growth.
Lateral and adventitious root
development ,
signal transduction
R (+) Maize R(+),L(−) (White
Lupin)
Hsieh et
al,2009
164 NAC transcription
factors
Accelerate senescence,
N remobilization
L(+) R(-)
S(-)
Maize R(+) SM(-)
L(-)
White lupin Liang et al
,2012
miRNA
family
Target gene
or protein
Description of
function
Involvement
under low N
Involvement
under low Pi
Referen
ce

169 HAP2 transcription
factors, CAAT binding
factor/NFYA
Nitrogen homeostasis, stress response
Nitrogen homeostasis, N uptake
Antioxidant
R(-) S(-)
L(-)
Maize SD(-)
R(-) S(-)
Arabido
psis
172 AP2 like transcription
factors
Ethylene-responsive pathway,
N remobilization ,Flower development
L(+) S(+)
R(-)
Maize
Arabid
opsis
L(-) Tomato Zhu et al
,2010
395 ATP sulfurylase; sulfate
transporters
Sulfate homeostasis R(-) Arabid
opsis
R(-) Arabido
psis
Zeng et
al,2010
397 Laccases Laccases Reduce root growth,
Copper homeostasis
L(-) R(-)
S(-)
Maize L(-) White
Lupin
Zhao et
al,2013
399 Ubiquitin conjugase
E2/UBC24
Phosphate homeostasis, uptake and
translocation
L(-) R(-) Maize R(+)
S(+)
SD(+)
Arabido
psis
Wang et
al,2013
Contd...

780 Na+/H+ antiporter Sodium ion export R(+) Arabid
opsis
Valdes et
al,2008
827 Ubiquitin E3 ligase with
RING and SPX
Nitrogen/phosphorus metabolism
Accelerate leaf senescence,
P homeostasis, P uptake
R(-)
L(-)
R(-)
Arabid
opsis
Maize
SD(+)
R(+)
R(+)
S(+)
Arabido
psis
Rice
Hackenberg
et al,2013
444 MADS-box Root development R(+) Rice R(+) Rice Lundmark et
al,2010
398 COX5b-1; CCS1 COX Copper homeostasis, oxidative stress
Enhanced to produce ATP under stress
R(-)
SD (-)
Arabid
opsis
SD(-)
R(-) S(-)
Arabido
psis
Xu et al,2011
171 SCARECROW-like
transcription factors
Root development R(+) Arabid
opsis
SM(+)
L(+)
White
lupin
Pant et
al,2012
Contd…

Role of miRNA in Nitrogen use efficiency
miR156 and Squamosa Promoter Binding Protein Like
(SPL)
SPL proteins play critical roles in maintaining normal growth throughout
plant life cycle .
These miRNAs are involved in phase change via their targets, members of
the SPL transcription factors.
miR156 targets, SPL3, is downregulated , suggesting that a miR156/SPL3
module might act by repressing vegetative phase change under limiting N
availability.
miR156 acts as a negative regulator of miR172 by controlling miR172
expression via its targets SPL9 and SPL15.

miR160 and Auxin Response Factor 16 (ARF16)
ARFs are DNA binding proteins that control auxin -regulated
transcription and are only present in plants.
ARF16, being this auxin responsive factor already characterized to
control root cap formation.
N-deficiency induces expression of miR160, which increases
ARF16 degradation and consequently supports lateral root formation.
miR169 and Nuclear Factor Y Subunit A (NFYA)
NFYA also called heme -activated protein (HAP) or CCAAT- box binding-factor(CBF),
NFYA have being associated with nodule differentiation and drought tolerance.
Transgenic Arabidopsis plants over expressing miR169a ,they showed a decrease in N
accumulation .These plants showed higher sensitivity to N limitation comparing to the wild
type, since NFYA regulates the nitrate transporters NRT1 and NRT2(Zhao etal.,2011) .

miR393 and Auxin Signaling F-Box Protein 3 (AFB3)
Nitrate is able to transcriptionally induce expression of AFB3 in roots and that N
metabolites produced after nitrate reduction and assimilation lead to a down regulation of
AFB3 levels due to miR393 induction .
miR393/ARF3 is the responsible mechanism to repress primary root elongation and
induce lateral root emergence under the presence of N.
The monocot specific miR444 has been demonstrated to regulate four MIKC-type
MADS-box transcriptional factor genes in rice (OsMADS23 , OsMADS27a , OsMADS27b
, and OsMADS57 .
miR444 targets with Arabidopsis ANR1clade, which is a pivotal regulator in NO3−
signaling pathway in lateral root growth.
Plants overexpressing this miRNA presented a decrease in the expression of the four
MADS- box genes, and a reduced nitrate induced lateral root growth .
miR444 and MADS-Box Transcription Factor

Kulchiski et al,2015Figure : The miRNA regulatory network in N signaling

Role of miRNA in P use Efficiency
The uptake and assimilation of P in plants is orchestrated by two key proteins , phosphate
transporter1 (PHT1) and phosphate 1 (PHO1).
PHT1 proteins are able to use energy to co-transport Pi and H+ and are therefore involved in Pi
acquisition .
PHO1, is involved in the loading of acquired Pi into xylem, facilitating therefore the root-to-shoot
transport of this macronutrient in plant.
Upon Pi stress , plants trigger Pi Starvation Responses (PSR). Since,PHT1 and PHO1 proteins are
central in the assimilation and allocation of Pi in plants, PSR try to maximize their expression .
The MYB transcription factors Phosphate Starvation Regulator 1 (PHR1) and Phosphate Starvation
Regulator 1- like (PHR1-LIKE1) leads to PHT1 and PHO1 over-accumulation.
 PHR1 and PHR1- LIKE1 induce the expression of Phosphate Transporter Traffic Facilitator 1
(PHF1), a protein that facilitates the transport of PHT1 to membranes, increasing therefore their
availability for Pi assimilation.
miR399, miR 827/PHT1,PHO1,PHO2

An E2 ligase, PHO2 mediates the
ubiquitination of the Pi/H+ transporters during
normal Pi conditions ,preventing their
trafficking to membranes.
PHO2 also mediates a post-translational
inhibition of PHO1 .Therefore, by repressing
PHO2, miR399 contributes to the
accumulation of both PHT1 and PHO1 during
Pi starvation .
PHT1 may also suffer ubiquitination by the
Nitrogen Limitation Adaptation(NLA) protein
, an E3 RING ubiquitin enzyme. NLA-
mediated ubiquitination of PHT1 leads to
endocytosis and degradation of the protein.
 The post-transcriptional regulation of the
NLA encoding gene by miR827 , therefore ,
helps to stabilize PHT1 levels during stress
conditions.
Figure: Hypothetical model for the role of miR399
& miR827 to maintain Pi homeostasis in plants.
Nguyen et al,2015

Figure: The miR399 and 827 pathway involve in P deficiency
Kulcheski et al,2015

Role of miRNA in K Homeostasis
MADS-box genes encode a family of transcription factors and are associated with several
developmental regulatory pathways, from root to flower and fruit development .
The unique miRNA investigated with respect to K+ signaling is the monocots specific
miR444a .The expression profile of this miRNA and its respective targets(MADS-
23,MADS-27a,MADS-27b,and MADS-57) during K+ deprivation in rice roots was explored
and it was found that this condition caused as lightly decrease of miR444a levels .
MADS-23 target was strongly induced compared to the control situation.
The presence of NH4+ in potassium containing medium favors nitrogen uptake by HAK5
protein , which is suggested to be a high affinity K+ transporter . Under this nutrition
circumstance ,the protein AKT1 turns to be the main K+ uptake protein even in low K+
concentrations .

Figure :Proposed models of regulation of K+ transportation by miRNAs
Kulcheski et al,2015

Role of miRNA in S homeostasis
In Arabidopsis, sulfate is transported through xylem or phloem via cell-specific transporters
such as sulfate transporters1;1 (SULTR1;1),SULTR2;1,andSULTR2;2.
sulfate limitation induces the expression of miR395 and its low affinity sulfate transporter
SULTR2;1,
restriction of SULTR2;1 expression by miR395 in the xylem parenchyma facilitates the
translocation of sulfate ions from the roots to the shoots.
miR395 has also been elucidated in S assimilation by sup- pressing the expression of ATP
sulfurylase genes, such as APS1, APS3, and APS4, which catalyze the first step of S
assimilation.
S deficiency leads to the elevated synthesis of SULFUR LIMITATION1 (SLIM1) protein in
the roots, which in turn activates various sulfate transporters to enhance S uptake.

Figure: miRNA mediated regulation of Phosphate & Sulphate
uptake , transport and assimilation in plants Kumar et al,2017

Role of miRNA in Copper homeostasis
Cu plays an important role against oxidative stress responses by acting as a
cofactor of Copper/Zinc superoxide dismutase (CSD).
superoxidedismutase SOD1 (CCS1) 1 is a chaperone protein that delivers the Cu
ions to CSD1 and CSD2 apoprotein .
During Cu limitation , the induction of miR398 downregulates ,SOD1 (CCS1)
gene for the expression of CSD1 and CSD 2 protein.
miR397, miR408, andmiR857, have been found to be up regulated during Cu
starvation, which in turn suppresses the expression of laccase and plastocyanin
genes.

Figure: miRNA mediated regulation of Cu homeostasis
Paul et al ,2015

CASE STUDY - 1
Material and Methods : Arabidopsis thaliana (ecotype Columbia) seeds were used in
this study. Sterilized seeds were suspended in 0.1% agarose and plated on MS
medium.

To characterize the small RNAs that are
responsive to C, N, or S deficiency, 10-day-
old seedlings grown on full nutrient (FN),
sucrose-free (–C), nitrogen-free (–N), or
sulfate-free (–S) MS medium were used to
construct small RNA libraries.
Figure: Response of seedling to Nutrient
Deficiency
By sequencing they identified the most
abundant size of small RNAs in the FN library
and –N Library was 21 nucleotides . In
contrast, 20 and 24-nucleotide-length small
RNAs were the most abundant in –C and –S,
respectively . These results suggested that
different nutrient supplies caused differential
distribution patterns of the sizes of small
RNAs.

Figure: Response of small RNAs to Nutrient deficiency

ICAR-IndianAgriculturalResearchInstitute,NewDelhi To explore the miRNAs that were differentially expressed in response to a specific nutrient deficiency, They
compared the read counts of miRNAs under nutrient-deficient conditions with those under FN. 92, 79,
and 59 differentially expressed miRNAs, which are clustered into 40, 41 and 31 miRNA families, were
obtained for –C, –N, and –S, respectively.

To investigate the putative functions of these specifically nutrient-responsive miRNAs , they analyzed their
target genes .

The responses of these miRNAs to various nutrient deficiencies were further confirmed by real-time PCR
Figure: Expressions of miRNAs and their Targets in response to Nutrient Deficiency

In addition, some miRNAs were positively
correlated with their targets, such as
miR395-APS3 in –S, miR397-LAC2 in –C,
and miR160-ARF17 in –C and –S. It is
likely that these targets are also regulated
by other transcription factors or their
expression does not completely overlap
with miRNAs Spatiotemporally.
Figure : Phenotype of Transgenic plants under the
Nutrient starvation conditions
Conclusion: A large number of miRNAs were differentially expressed in response to nutrient deficiencies,
some miRNAs were specifically responsive to specific nutrient depletions. miR169b/c, miR826, and miR395
showed the largest changes in response to –C, –N, and –S, respectively.
To investigate the functions of nutrient-
responsive miRNAs in nutrient starvation
adaptation, miRNA overexpression plants
(miR160a-ox, miR395a-ox, and miR399b-
ox) and miR160 suppression plants
(STTM160) were used to evaluate
phenotypes in nutrient starvation conditions.

Case study-2
 Polymorphism of flax (Linum usitatissimum L.) genome, genotype CDC Bethune, under nutrient stress in
vitro, was analyzed by newly developed type of molecular markers based on microRNA molecules.
Two types of stress-sensitive miRNAs , miR395 and miR399 were evaluated. The miR395 loci profile has
shown to be more polymorphic and more specific in comparison to miR399 loci pattern.
They supported the role of miRNA molecules as potential biomarkers of abiotic stress.

 Flax is known by its phytoremediation capabilities . In addition, flax is of research interest
because some lines undergo phenotype and genome changes in response to environmental
conditions (Cullis 2004).
 They applied nutritional stress under in vitro conditions on the genotype CDC Bethune &
were interested at the level of miRNA-based markers, to record the polymorphism of nutrition
stress-sensitive miRNA, miR395 and miR399, in the genome of flax.
Seeds of flax genotype CDC Bethune were cultivated on four different nutritional variants of
the MS basal medium as follows:
1 – full-strength of microelements and vitamins, half-strength of macroelements,
2 – full-strength of macroelements and vitamins, half-strength of microelements,
3 – full-strength of microelements and macroelements, half-strength of vitamins,
4 – half-strength of microelements, macroelements and vitamins,
C – control variant – basal MS medium.
In vitro cultivation was carried out during the period of six weeks at 22°C under photoperiod
16 h light/8 h dark cycle & then Total genomic DNA was extracted .

The primers for the miRNA-based markers were designed according to the mature
miRNAs sequences, originated from the miRNA database (http://www.mirbase.org/) .
A total of 2 miRNA-based forward primers and 1 universal miRNA reverse primer
were used and randomly combined together to perform a marker assay.
Combination of primer pairs used for miRNA-based marker assay.

They found variability not in no. of miRNA Loci
but also in their size.
highest number of miR395 loci (9) has been
recorded at the MS basal medium having half-
strength of macroelements .
 Within the macroelements of MS medium
sulfur is present only in one component and that is
magnesium sulphate while, four microelements
are in the form of sulphates (copper, iron,
manganese and zinc).
Figure : Representative gel showing amplification profiles of
CDC Bethune generated by primer pair miR 395_F / miR_R.
Their results supports the possibility that
miR395 response varies between different
plant species and some plants that are
adapted to inferior growing conditions
might evolved constitutive adaptive
mechanisms.

ICAR-IndianAgriculturalResearchInstitute,NewDelhi The pattern of miR399 is less polymorphic than the
miR395 pattern.
 They observed significantly different miR399 loci
pattern in the case of stress variant having half-
strength of all components of MS medium.
 Within the macroelements of MS medium
phosphate is present only in one component and that
is potassium phosphate- while in the microelements
there are no phosphates.
Figure: Representative gel showing amplification profiles of
CDC Bethune generated by primer pair miR 399_F / miR_R.
miRNA based molecular markers are sufficient for evaluation of flax genome polymorphism under specific
condition of abiotic stress . It shows the capability of miRNA molecule as potential biomarkers of
environmental stress.
 That means that the stress variant number 1
represents, in this case, the conditions of low
phosphate characterized by miR399 up-regulation.
In this variant the highest number of miRNA loci in
comparison to control has been observed.

Conclusion
The alteration of nutrient levels in soil can trigger specific signaling molecules that can act as
repressors of target nutrient responsive- miRNAs . The decreased accumulation of miRNAs
subsequently stabilizes the expression of transporters .
majority of miRNAs involved in NPK deprivation are associated with mechanisms involved
in the adaption to stress conditions by affecting root architecture ,controllingNO3− or Pi
transporters , controlling shoot growth, affecting vegetative phase transition , and managing
these nutrients leakage.
On contrary, the optimal conditions or higher amounts of nutrients can trigger a specific
group of miRNAs that directly affect the transporter or induce other miRNA s that suppress the
expression of repressor genes.

Future Prospective
Investigation of novel miRNAs and their role in phytate biosynthesis like regulation of
different inositol phosphate kinase genes, alteration of specific miRNA expression by
overexpression or genome editing.
Role of miRNAs in regulation of nitrate transporters and metabolic enzymes such as aspartate
amino transferase, glutamine synthase, glutamate dehydrogenase .
Role of miRNAs in different groups of Fe and Zn transporters from roots to seed, miRNA
promoter/ genome editing .
Identification of root specific novel miRNAs under nutrient stress and investigation the role of
miRNA- mediated miRNA activation or removal of supressors of transporters .
Role of miRNAs in down-regulation of heavy metal transporters .

Role of miRNA for Nutrient Use Efficiency in crops

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