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Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency. 組員 : 彭元慶 林柏齡 郭宇翔 陳傑君 . Author : Surya Kant, Yong-Mei Bi and Steven J. Rothstein. Content. Introduction

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Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency

組員: 彭元慶




Author : Surya Kant, Yong-Mei Bi and

Steven J. Rothstein

  • Introduction
  • Physiological and molecular componentsgoverning NUE
  • Understanding plant response to N limitation with physiological and molecular approach
  • Future prospect
need for genetic improvement of nitrogen use efficiency nue
Need for genetic improvement of nitrogen use efficiency (NUE)
  • Population growth
  • Cost of N input


Marginal effect of N use

N waste

  • Genetic affect

definition of nitrogen use efficiency nue
definition ofnitrogen use efficiency (NUE)

Yield per unit of N available in the soil

Allen et al., 2004

physiological and molecular components governing nue
Physiological and molecular componentsgoverning NUE
  • Two general stages for N use in the plant life cycle
  • Vegetative stage N uptake and assimilation
  • Reproductive stage N remobilization

Chardon et al., 2012

uptake and assimilation
uptake and assimilation
  • Nitrate transport (NRT):In Arabidopsis, three families of nitrate transporters NRT1, NRT2, and chloride channel (CLC )with 53 NRT1, 7 NRT2, and7 CLC genes are identified.
  • The ammonium derived from nitrate or from direct ammonium uptake by Ammonium transporters(AMT)

NRT1.1, NRT1.2, NRT2.1, andNRT2.2


Markus et al..2009

Xuet al., 2011

  • NRT1.1 is adualaffinitynitrate transporter

NRT1.1 is a dualaffinitynitrate transporter

Markus et al..2009

chl1 functions as a nitrate sensor in plants
CHL1 Functions as aNitrate Sensor in Plants

Cheng-Hsun Ho, Shan-Hua Lin, Heng-Cheng Hu, and Yi-Fang Tsay


n uptake supplement
N uptake(supplement)
  • Nitrate&ammonium之間的調控與平衡

Anthony D.M. et al.,2002


Regulation of



Extemal input

(Grey )



Markus et al..2009

n assimilation
N assimilation
  • Glutamine synthetase

cytosol (GS1)

plastids (GS2)





In maize led to an increase of 30% in kernel number

(Martinet al., 2006)

Cytosolic GS1

Enhanced N accumulation capacity

in shoots and grainsinwheat

Jed P. Sparks 2009

n remobilization
N remobilization
  • Step1: The protein degradation

1.Chloroplast degradation pathway

2.Vacuolar and autophagic pathway

3.Ubiquitin-26S proteasome pathway

  • Step2: Amino acid remobilizing
vacuolar and autophagic pathway
Vacuolar and autophagic pathway

D. E. Martı´nez,2008

Sigrun et al., 2010

ubiquitin 26s proteasome pathway
Ubiquitin-26S proteasome pathway

Lui et al., 2008

n remobilization1
N remobilization
  • After proteindegradation

all the amino acids are remobilized, glutamine, asparagine followed by glutamate, aspartate, serine, and alanine are predominant in

phloem sieve tube

amino acid permeases aap
Amino acid permeases (AAP)
  • lysine/histidine transporter (LHT)
  • cationic amino acid transporters (CAT)
  • proline transporters (ProT)
  • aromatic and neutral amino acid (ANT)
  • transporters (ANT)
  • oligopeptide transporters (OPT)
n remobilization2
N remobilization
  • Much evidence supports the role of cytosolic

GS1 in the efficient remobilization of amino acids forsenescing leaves towards grain-filling

morphological response of root system to n supply
Morphological response of root system to N supply
  • Signal for development processes
  • genetic and environmental factors
signal for development processes
Signal for development processes
  • N concentration affects root development, root architecture, and the root-to-shoot ratio
  • low N supply generally leads to decreased root growth, suppression of lateral root initiation, increase in the C/N ratio within the plant, reduction in photosynthesis, and early leaf senescence
signal for development processes1
Signal for development processes
  • TheANR1 dual regulation of lateral roots by nitrate is developmental stage dependent

(Zhang and Forde, 1998, 2000; Zhang et al., 1999).

  • NRT2.1acts as a positive regulator for lateral

root initiation under N limitation conditions (Remans et al. 2006b)

  • By contrast, reported a repressive role in lateral root initiation (Little et al. 2005)
plant s adaptive response to n limition
Plant’s adaptive response to N limition
  • Increase in N uptake by high affinity transporters
  • Remobilization of N from older to younger leaves and reproductive part
  • Retardation of growth and photosynthesis.
  • Increased anthocyanin accumulation.
understanding plant responseto n limitation
Understanding plant responseto N limitation
  • 1.Transcriptional profiling
  • 2.Reverse genetics
  • 3.Forward genetics
  • 4. Another model plant: Thellungiellahalophila(鹽芥)
transcriptional profiling
Transcriptional profiling:
  • Microarray
  • Sequence-based transcription profiling technology
  • next-generation sequencing approaches
reverse genetics
Reverse genetics
  • 利用T-DNA 插入正常Arabidopsis某gene片段
  • 形成失去某功能的nla mutant
  • 比較nlatype 和 wild type 在低N環境(3 mM)和正常N環境(10 mM)的生理型態、轉錄程度(whole genome transcript profiling,及生化機制(ex:phenylpropanoid pathway )的不同
  • 正常N環境下兩者無明顯差異;低氮環境下有1,272個基因調節方式不同,其中807個 up-regulated,465個down-regulated (Penget al.,2007a).


reverse genetics nla mutant
Reverse genetics: nla mutant

(Peng et al.,2007a)

reverse genetics1
Reverse genetics
  • In nla mutant plants, the phenylpropanoid pathway wasdisrupted, with substrates from this pathway channelledtowards lignin production and thereby anthocyanin synthesiswas suppressed.

(Penget al.,2008)

reverse genetics2
Reverse genetics

(Penget al.,2008)

reverse genetics3
Reverse genetics

(Penget al.,2008)

reverse genetics4
Reverse genetics

Pi limitation induced anthocyanin accumulation and

prevented the N limitation-induced early senescence

phenotype in the nla mutant

(Penget al.,2008)

reverse genetics5
Reverse genetics
  • NLAmight be controlled bya micro-RNA (miR827) and is an important component forthe integration of phosphorus- and N-limitation responses.

(Pant et al.,2009)

  • NLA gene also has a role in immune responses but as a negative regulator for salicylic acid production (Yaeno and Iba, 2008)
forward genetics
Forward genetics
  • 利用reporter gene 和 activation tagged lines 判斷係由何種組成影響N-limitationresponse.



Reporter gene



forward genetics1
Forward genetics
  • Ex:Girin et al. (2010) used transgenic Arabidopsis plants harbouring a NRT2.1 promoter::LUC reporter gene to screen EMS mutagenized plants and have identified three mutants that appear to be altered in their regulation of nitrate uptake.
forward genetics2
Forward genetics

阿拉伯芥 (pNRT2.1::LUC)


( Cto T )





低Ninduce LUC gene 轉譯Luciferase


三個hni mutant:




Luciferase(LUC) 與luciferin反應呈螢光

(Girin et al. 2010)

NRT2.1 promoter

LUC gene

forward genetics3
Forward genetics

Figure 1. Characterization of NL plants (pNRT2.1::LUC). Plants were grown on vertical agarose plates for 7 d on HN or LN medium supplemented with 1% Suc. A, Bioluminescence imaging and quantification of the LUC activity (values are means of six replicates ± SD).

B, Relative accumulation of the LUC transcript (values are means of three replicates ± SD).

C, Relative accumulation of the NRT2.1 transcript(values are means of three replicates ± SD).

D, High-affinity influx measured in 0.2 mM 15NO3 - (values are means of 10 replicates ± SD).

DW, Dry weight.

forward genetics4
Forward genetics

Figure 2. Characterization of NRT2.1 expression in hni mutants. Plants

were grown on vertical agarose plates supplemented with 1% Suc for

7 d. A, LUC activity in hni mutants and NL in plants cultivated on HN

or LN medium. Values are means of six replicates 6 SD. B, Relative

accumulation of the NRT2.1 transcript on HN or LN medium. Values

are means of three replicates 6 SD.

(Girinet al. 2010)

another model plant thellungiella halophila
Another model plant:Thellungiellahalophila(鹽芥)

(Inanet al. 2004)

  • Comparison with 阿拉伯芥
  • 92%序列相似
  • small genomesize: 2倍
  • short life cycle :6至8星期
  • copious seed number:6,000–8,000
  • Ease of transformation
another model plant
Another model plant:

低N環境下,鹽芥 could maintainhigher

N content,

total amino acids

total solubleprotein

低C/N ratio

by efficiently acquiring and utilizing nitrate .

(Kant et al., 2008).

another model plant1
Another model plant:

white bars, 4 mM nitrate;

light gray bars, 1 mM nitrate;

dark gray bars, 0.4 mMnitrate

(Kant et al., 2008).

another model plant thellungiella halophila1
Another model plant:Thellungiellahalophila(鹽芥)
  • The production of BAC and cDNA libraries, and the generation

of EST and T-DNA insertion collections will further

enhance the power of the Thellungiella system for identifying

key components governing N utilization under low N conditions.

candidate gene for improving nue
Candidate gene for improving NUE
  • Function in the transport of ammonium or nitrate
  • Regulatory function (ex: some transcription factor genes)
  • Early nodulin gene( completely unknown function)
candidate gene gata transcription factor genes
Candidate gene : GATA transcription factor genes
  • GATA transcription factor genes regulate N assimilation in plants.

(Jarai et al., 1992; Rastogi et al., 1997; Oliveira and Coruzzi, 1999).

  • GATA factors play a role in NiR gene regulation

(Rastogi et al., 1997).

candidate gene gata transcription factor genes1
Candidate gene : GATA transcription factor genes
  • GNC(GATA, nitrate-inducible, carbon metabolism-involved).gene is one member of the GATA transcriptional factor gene family.
  • STP13(Sugar transport protein)is one of the C metabolic genes whose expression levels were tightly influenced by GNC (Bi et al., 2005).
candidate gene gata transcription factor genes2
Candidate gene : GATA transcription factor genes

55 mM glucose and

varying nitrate concentrations

Ox:STPoverexpressor line

(Bi et al., 2005).


candidate gene early nodulin gene unknown function
Candidate gene : Early nodulin gene( unknown function)
  • The transgenic plants over-express early nodulin gene (OsENOD93-1) had a significant 10–20%mincrease in the number of spikes and spikelets, and seed yield under both limiting-N and optimum-N conditions, and a significantly higher shoot dry biomass than wild-type plants under limiting-N conditions (Bi et al., 2009)

sequence homology of 58.2% to GmENOD93

( Reddy et al.,1998)

candidate gene early nodulin gene unknown function2
Candidate gene : Early nodulin gene( unknown function)
  • ENOD40 has been shown to be expressed during the earlystage of nodule initiation (Kouchi & Hata 1993;Yang et al.1993), but its protein product may be involved in corticalcell division (Charonet al. 1997), differentiation of vascular

bundles (Kouchi et al. 1999) or photosynthate transport(Kouchi & Hata 1993; Yang et al. 1993). A number ofhomologs of ENOD genes have been identified in nonleguminousplants (Trevaskiset al. 1997; Reddy, Kouchi &Ladha 1998), but most are of unknown function

  • there are many potential genetic approaches for the improvement of crop NUE and these require increased knowledge of how plants respond to different N regimes as well as to other environmental conditions.
referance paper
  • Exploring NUE in crops and in Arabidopsis ideotypes to improve yield and seed quality

Xuet al., 2011

  • Exploring NUE in crops and in Arabidopsis ideotypes to improve yield and seed quality

Chardon et al., 2012

  • Can less yield more? Is reducingnutrient input into the environmentcompatible with maintaining cropproduction?

Allen al., 2004

  • Molecular and Developmental Biology of Inorganic Nitrogen Nutrition

Nigel M. et al 2002

  • Protein Degradation and Nitrogen Remobilization during Leaf Senescence

Lui et al., 2008

referance paper1
  • The nitrate/proton antiporterAtCLCa mediates

nitrate accumulation in plant vacuoles

A. De Angeliet al., 2006

  • Ecological ramifications of the direct foliar uptake of nitrogen

Jed P. 2009

  • The regulation of nitrate and ammonium transport systemsin plants

Anthony D.M. et al.,2002

  • From signal transduction to autophagy of plant cell

organelles: lessons from yeast and mammals

and plant-specific features

Reumannet al.,2010

referance book
Referance - book
  • Transporters and Pumps in Plant Signaling

Markus Geisler and KeesVenema 2009