此文为傅向东老师在水稻GA信号和氮素信号的又一经典之作，值得我们好好学习，包括研究的思路、文字的写作、图表制作等各个方面！下面仅仅对文章正文中重要的表达进行摘录学习，后面还会有更深入的学习！
1XX是XX的基础
because environmentally degrading inorganicfertilizer use underlies current worldwide cereal yields
重点单词的使用
enhanced nitrogen use efficiency
facilitates recruitment of XX to repress XXvia XX
competitively inhibit the interaction andexplain increased XX varieties
in contrast
in part
due to
enhance cereal crop yield
enhance the activity of growth-repressingDELLA proteins
a growing world population
green revolution varieties
be conferred by
XX allele conferred similar properities
mutant alleles
stimulate the destruction of DELLAs
promote plant growth
resist destruction
be resistant to lodging
reduces gibberellin abundance
increase accumulation of the rice DELLAprotein
the flattening of plants by wind and rain
green revolution rice varieties
require
be require for
be responsible for
account for
a high-nitrogen fertilizer supply
achieve maximum yield potential
the drive toward increased agriculturalsustainability
必需
necessitate reduced nitrogen fertilizer use
grain yield
a sum of the multiplicative integration ofthree major components
tiller numbers per plant, grain numbers perpanicle, and 1000-grain weight
an increased tillering ability
high-density planting conditions
contributes to
the high-yield properties of XX
a key cereal breeding goal
define the mechanisms underlying thepromotive effects of nitrogen on tiller bud outgrowth
show how genetic modulation of thesemechanisms can enable increased grain yield of green revolution varietiesdespite reduced nitrogen input, thus advancing agricultural sustainability.
nitrogen promotes rice tillering via NGR5
found that
increased with increasing nitrogen supply
additional effects of increased nitrogen
increases in grain number and yield
display nitrogen-dependent tiller numberincrease
under different nitrogen fertilizationlevels
with tiller numbers being consistentlyhigher in XX than in SS
termed XX, named XX
in contrast, either exogenous gibberellintreatment or overexpression of the rice bibberellin receptor GID1, inhibitednitrogen-promoted tillering.
thus
Thus, the enhanced DELLA function typicalof green revolution varieties increases nitrogen-induced promotion of tillernumber.
further analysis showed that
elite
increases in xx in xx variety were not dueto
initiating outgrowth and tiller branchextension
screen
an ethy methane sulfonate-mutagenized 9311population
display
an altered tiller number nitrogen response
among such mutants
display a reduced tiller number
be sensitive to changes in nitrogen supply
map-based cloning and geneticcomplementation
reveal
encode an XX transcription factor
identifying an unknown function for NGR5 innitrogen-responsive tillering regulation.
carry a XX substitution conferring a mutantprotein
which fails to complement ngr5 phenotype
in addition to its effect on tiller number
be required for
accordingly
whereas
grain yield per plot increasedprogressively with increasing nitrogen supply, this effect was abolished inngr5 plants
further analysis showed that
lack of NGR5 had no effect on the formationof tiller buds ( lateral bud initials), but reduced the number initiatinglateral outgrowth and tiller branch extension.
confirming that nitrogen-responsiveregulation of tillering is dependent on NGR5
be dependent on NGR5
an increasing nitrogen supply increasedNGR5 abundance at both mRNA and protein levels
be abolished
had no effect on
accumulation of NGR5-HA fusion proteinincreased with increasing nitrogen supply
furthermore
positively regulate
over a wide expression range
p35S::NGR5 transgene
mimicking the effect of
on tillering capacity
we conclude
promotes increased NGR5 abundance, which inturn promotes tiller bud outgrowth
suppressed
贡献
confer
be necessary for
characteristic for green revolutionvarieties
ngr5 represses branching-inhibitory gene
抑制
repress, inhibite, suppress,
诱导
induce, promote，
提高
enhance，increase,
减小
reduce, decrease
揭示观点
imply, define，display, reveal,suggest
缺少
lack of XX
导致
cause, result in, lead to,
multiple differentially expressed genes displayingan increase in XX
gene set enrichment analysis
a correlation between XX and XX
genes up-regulated in XX
the set of XX-marked genes already known tobe normally repressed by histone modification
with marks occurring at both TSS and genebody regions of ngr5-up-regulated genes
suggests
参与
be involved in
repression
identified, cloned, characterize
the receptor for
the XX component of XX
targets XX repressor
proteasomal destruction
encoding a TCP domain transcription factor
be already known to inhibite lateralbranching and tiller number.
high nitrogen supply reduced the abundancesof mRNA specified by these shoot branching-inhibitory genes, and this effectwas abolished by lack of NGR5 function.
lack of D14 or OsSPL14 function isepistatic to ngr5 in regulating lateral branching.
thus, D14 and OsSPL14 function downstreamof NGR5, and NRG5 mediates nitrogen-promoted increase in tiller number byrepressing the inhibitory functions of D14 and OsSPL14 branching-regulatorygenes
ChIP-PCR experiments revealed
furthermore
the extent and effect of
correlated with
mRNA abundance decreased with increasingnitrogen supply
the extent of NGR5 binding and the level ofXX modification at D14 were correspondingly increased in a nitrogen-dependentmanner.
similar effects were observed for OsSPL14,which suggests that NGR5 promotes tillering in response to increasing nitrogensupply by binding to target branching-inhibitory genes, thus causing theirrepression through regulation of H3K27me3 modification
NGR5 recruits PRC2 for H3K27me3 deposition
determine how NGR5 regulates regulatesnitrogen-promoted H3K27me3 modification
perform a yeast two-hybrid screen for NGR5interactors,
identifying
interactions be confirmed in BiFC and Co-IPexperiments
furthermore, a CROSPR/Cas9-generated LC2reduced-function allele was shown, to abolish
lc2 also suppressed the increased tillernumber conferred by XX
lack of XX function was epistatic to lc2
Taken together
suggest that
depends on LC2 function
conducted XX surveys of XX methylation inresponse to varying nitrogen supply
increasing nitrogen supply altered XXpattern, the extent of this alteration was reduced
in ngr5, which suggests thatnitrogen-mediated genome-wide reprogramming of XX methylation
is NGR5-dependent.
we next performed xx experiments andidentified a total of 453 binding shared in common by XX and XX
further analysis identified potential target-siterecognition motifs shared by XX and XX, with a predominant shared GCCGCC motifbeing common in the gene body regions of both XX and XX
reduced D14 mRNA abundance in wild-typeplants, and these effects were abolished in lc2 plants, just as they were inngr5 plants.
Furthermore, increasing nitrogen supplyprogressively increased XX
these observations suggest that NGR5-drivenrecruitment of X to X results in repressive modification of these genes inresponse to increased nitrogen supply, thereby promoting bud outgrowth andincreasing till number
NGR5 is a target of gibberellin receptorGID1
as show above, nitrogen-induced increase intiller number was enhanced in green revolution varieties, and this effect wasinhibited by exogenous gibberellin treatment.
analysis of both RNA-seq and ChIPsequencing revealed multiple common gene targets to be co-regulated by NGR5 andGA treatment.
furthermore, GA treatment altered thechange in genome-wide XX modification patterndue to
increasing nitrogen supply in a mannersimilar to the alterationconferred by ngr5,whereas a partially restored XX modification pattern was induced by treatmentwith PAC, an inhibitor of gibberellin biosynthesis.
表示的意思是GA处理能改变甲基化修饰（甲基化降低），在增加N高氮供应的情况下已一种方式。这个方式和有ngr5的改变很相似，然而使用PAC处理能够恢复这些改变。
简要意思就是在N增加情况下，甲基化升高，但在高氮情况下，GA处理甲基化下降，这和ngr5一样。
inhibit the nitrogen-dependent increase in XXmodification and consequent repression of expression of shoot branchinginhibitor genes.
this observations suggest the existence ofa mechanistic link between N- and GA-mediated effects on tiller number
in canonical gibberellin signaling, GAbinds its receptors GID1,thus recruiting DELLAs for ployubiquitination by theF-box protein XX and subsequent destruction in the 26S proteasome, thuspromoting plant growth.
reduced xx function in XX mutant led to anincreased tiller number above that of NJ6 controls in both high and lownitrogen supply
conversely, plants overexpressing GID1under the control of the 35S promoter exhibited nitrogen-insensitive responses,with lower tiller number than in nontransgenic controls.
Although gibberellin repressed the tillernumber in both NJ6 and NJ6-sd1 plants, it had no effect or tiller number in xxplants, nor in XX plants overexpressing.
furthermore, either gibberellin-inducedinhibition or GID1-mediated repression of tillering mimicked the effect ofngr5. XX和 XX 有相似的功能，可以用
similar effects can be achieved by overexpressingxx.
this results suggest thatgibberellin-GID1-mediated repression of till number is dependent on thenitrogen-regulated function of NGR5.
NGR5 abundance is negatively associatedwith gibberellin level:NGR5-HA accumulation was increased in relativelybiggerellin-deficient NJ6-sd1 plants (versus NJ6), whereas it was reduced byexogenous gibberellin treatment.
conversely a gibberellin-mediated decreasein NGR5-HA abundance was inhibited by treatment with the proteasome inhibitorMG132, such that NGR5-HA accumulation was increased above that of NJ6-sd1plants.
accordingly, western bolt analysis detectedthe accumulation of polyubiquitinated NGR5-HA in the presence of MG132, whichsuggests that gibberellin promotes polyubiquitination and subsequent proteolysisof NGR5 in the 26S proteasome.
in addition, gibberellin-induceddegradation of NGR5-HA was inhibited in theNJ6-gid1-10 mutant, indicating thatgibberellin-induced promotion of NGR5 polyubiquitination and proteasomedestruction is dependent on the GID1 function
gibberellin responses are conventionally
considered to be activated by GID1-mediated destruction of DELLAs. however,
gibberellin-mediated degradation of NGR5-HA occurs either in the absence of DELLAs
in a loss-of-function slr1 mutant or in the presence of the high-level DELLA
accumulation conferred by the slr1-d6 gain-of-function mutation. Although the
mutant SLR1, DELLA encoded by slr-d6 was relatively resistant to
gibberellin-mediated destruction, NGR5-HA was still destabilized by exogenous gibberellin
treatment. Thus, gibberellin-promoted destabilization of NGR5, although
dependent on GID1, is neither dependent on nor downstream of
gibberellin-induced destruction of DELLAs. 既不依赖DELLA，也不是GA诱导的DELLA降解的下游。
we therefore explore thepossibility of an alternative, previously unknown, DELLA-independent mechanismwhereby the E3 ubiquitin ligase directly mediates gibberellin-promoteddestruction of NGR5, finding that GID1 interacts directly with NGR5 (as assayedby bifc and co-IP) and that the strength of this interaction of thisinteractions is potentiated by increasing concentrations of gibberellin. thus,as with the DELLAs, gibberellin enhances the interaction between NGR5 and GID1,thereby identifying NGR5 as a potential alternative substrate for GID1-promotedpolyubiquitination.
although NGR5 lacks the specific DELLAmotif that enables the GID1-DELLA interaction, we found a motif within thedomain of NGR5 to enable the GID1-NGR5 interaction. furthermore, NGR5 alsointeracted with the GID2 F-box component of the E3 ubiquitin ligase thatnormally targets SLR1 for destruction in the 26S proteasome.
accordingly, an in vitro ubiquitinationassay show that GST-NGR5 fusion protein is polyubiquitinated by GID2-flagfusion protein in the presence of E1, E2, and ubiquitin, but not in the absenceof GID2-Flag, which suggests that NGR5 is a substrate of the SCF E3 ubiquitinligase.
additional time-course experiments showedthat gibberellin promotes the progressive degradation of GST-NGR5 but that thisdegradation is inhibited both by MG132 and in XX mutant. Finally, lack of GID2function also inhibits gibberellin-mediated regulation of NGR5 is not due togibberellin-promoted destruction of DELLAs, but is due to a previously unknowndirect and gibberellin-potentiated interaction of NGR5-GID1, leading topolyubiquitination of NGR5 by the E3 ubiquitin ligase and subsequentdestruction in the proteasome.
DELLA-NGR5 modulation of tiller N response
NGR5 interacts directly with SLR1 in yeasttwo-hybird screens in BiFC and Co-IP assays
nonetheless, we found that lc2-NGR5interaction is not inhibited by the presence of SLR1, which suggests that theSLR1-NGR5 interaction does not directly interfere with the lc2-ngr5 interactionthat determines NGR5 function. further experiments showed that the XX motif ofthe DELLA protein is necessary for the NGR5-SLR1 interaction. thus, in additionto both being substrates of XX, SLR1 and NGR5 interact directly with oneanother. with the XX motif being conserved in all the GRAS proteins, we nextfound that NGR5 interacts with two additional GRAS proteins previously shown toregulate tiller number.
the competitive nature of the SLR1-NGR5relationship with respect to GID1 caused accumulation of NGR5-HA to furtherincrease the accumulation of SLR1 in 9311.
determine whether competitive XXrelationships also GA and nitrogen effects on tiller number. as shown in Fig1b,the enhanced DELLA function conferred by XX allele, results in increased tillernumber. we therefore tested the possibility that the effect of XX on tillernumber might be due to differential effects on NGR5 stability. accordingly, theextent of the interaction between GID1 and NGR5 is reduced by the presence ofXX. we then confirmed the expectation that a reduced GID1-NGR5 interaction inthe presence of XX reduces the rate of XX destruction, and similar reductionsto be conferred by accumulation of wild-type XX proteins. further comparativestudies showed XX destruction to be more rapid that that of His-SLR1, thus,although gibberellin-promoted NGR5 destruction was DELLA-independent,competition NGR5 and SLR1 for GID1 interaction reduced the extent of NGR5-GID1interaction. moreover, the abundance of NGR5-GFP fusion protein was increasedwith PAC treatment but was reduced in response to combined GA and PACtreatments. this is also consistent with the observations that ngr5 exhibits ahigher ratio of GA-induced leaf sheath growth, whereas transgenic plantsoverexpressing NGR5-HA display reduced sensitivity to PAC treatment relative towild-type controls. we conclude that the enhanced DELLA function characteristicof both wheat and rice green revolution varieties competitively inhibits theGID1-NGR5 interaction, thus stabilizing NGR5 by reducing GA-GID1-mediateddestruction.
promotion of rice tilling isNGR5-dependent, we generate 9311NILs carrying various combinations of differentsd1, gid1, and ngr5 alleles. although the increased SLR1 accumulations in bothSS and ZZ increased the till numbers of plants grown in either low or high nitrogensupply (versus SD1), there was almost no difference in tiller number when XXand XX were compared. thus, della-mediated enhancement of nitrogen-inducedtiller number increase typical of green revolution rice varieties is dependenton NGR5 function. accordingly, comparisons of NGR5-regunated mRNA abundance andH3K27me3 modification of branching-inhibitory D14 and OsSPL14 genes in 9311versus 9311-SD1 revealed mRNA abundance and modification status at 0.6N in9311-SD1 to be roughly equivalent to that at 0.2N in 9311-sd1. thus, theenhanced DELLA function of sd1 increases tiller number in response to nitrogensupply by increasing the stability of NGR5, which in turn inhibits theexpression of shoot branching inhibitor genes, thereby promoting till number.
NGR5 improves yield and nitrogen useefficiency
determined whether an increase in NGR5abundance beyond that seen in elite rice varieties could further increasetiller number and yield in reduced nitrogen fertilizer input. first, wesurveyed publicly available rice varietal genome sequence data for naturalgenetic variation at NGR5, distinguished five distinct haplotypes, and foundthat H2 was associated with increased NGR5 mRNA abundances in both low and highnitrogen conditions, together with increases in tiller number and field-growngrain yield of 686 diverse Asian cultivated rice accessions. further analysisshowed that Hap-containing XX, one of the highest-yielding of indica varietiescultivated in china since the 1980s, displaying a greater NGR5 mRNA abundancethan did XX, and other lines, even at low and moderate nitrogen supply.
investigate, explore, known,
a transgenic mimic of H2 enhanced 9311grain yield in range of nitrogen supply conditions, without affecting thecharacteristic and beneficial semi-dwarfism of 9311. breeding with H2 is afeasible future strategy toward improving nitrogen use efficiency of the eliterice varieties. finally, having recently shown that allelic variation at GRF4enhances grain yield and nitrogen use efficiency through coordinating effectson carbon and nitrogen metabolic regulation. we investigated the geneticinteraction between XX and XX
increased abundances of both XX and XXfurther enhanced 9311 yield and nitrogen use efficiency, particularly atrelatively low levels of nitrogen supply.
nitrogen determines genome-wide chromatinstatus via NGR5-dependent recruitment of the XX complex PRC2 to target genes,among which are tiller branch-repressing genes.
in consequence, repression of tilleroutgrowth is reduced in increasing nitrogen supply, causing increasedtillering. we have also shown that NGR5 is a non-DELLA target XX and that XXcause the XX yield-enhancing tillering increases typical of green revolutionrice varieties.
because NGR5 is already known to beinvolved in the cross-talk between auxin and BR signaling. our discoveries addto a growing understanding of how diverse modes of molecular and functionalcross-talk between multiple phytohormonal signaling and fertilizer useresponses function in the environmentally adaptive regulation of plant growthand development. finally,we have shown that increasing NGR5 expression or
activity provides a breeding strategy to reduce nitrogen fertilizer use while
boosting grain yield above what is currently sustainably achievable.
