SYNTHETIC MICRO PROTEINS - VERSATILE TOOLS FOR THE REGULATION OF PROTEIN FUNCTIONS IN CROP PLANTS

DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
BHUBANESWAR - 751003
DOCTORAL SEMINAR - II
SYNTHETIC MICRO PROTEINS - VERSATILE TOOLS FOR THE
REGULATION OF PROTEIN FUNCTIONS IN CROP PLANTS
Presented By : -
Jyoti Prakash Sahoo
01ABT/Ph.D./17
JYOTI PRAKASH SAHOO, DEPT. OF AGRIL. BIOTECH. , OUAT, BBSR - 751003 1
Advisor : -
Dr. K. C. Samal (Professor)
Dept. of Agril. Biotech.
CA, OUAT, BBSR
30-07-2020

Microproteins
 MicroProteins are small proteins that contain only a single protein domain, often a
protein–protein interaction (PPI) domain but lack other functional domains.
 MicroProteins can either completely inactivate their targets by forming non-
functional heterodimers or alter their biological function by engaging the target
protein in novel protein complexes.
 The first identified microProtein, INHIBITOR OF DNA BINDING (Id) in animals.
It is a 16 kDa small protein consisting of only a helix–loop–helix (HLH) domain.
 LITTLE ZIPPER (ZPR) proteins were the first microProteins characterized in
plants. ZPR proteins contain a leucine zipper domain but lack other domains
required for DNA binding and transcriptional activation.
 ZPR proteins thus function in analogy to Id-type proteins and physically interact
with class III homeodomain-leucine zipper (HD-ZIPIII) transcription factors to
control developmental processes such as stem cell maintenance in shoot apical
meristem (SAM) formation and leaf development.
(Bhati et al., 2018)
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Different Modes of Microprotein Regulation
by interacting with ion channel
subunits and compromising
their transport capacity
by sequestering their
targets into non-
functional complexes
by attracting chromatin
repressor proteins (R)
by sequestering the target in
a subcellular compartment
where it is inactive
MIFs – MINI ZIP FINGERS
Flower Architecture
and Leaf Development
MIF2 and SIIMA
(Tomato Homolog) –
Inhibitor of Meristem
Interacts with TOPLESS
and HISTONE
DEACETYLASE19
Represses target
gene expression
1
2
Plant Specific miP1a and miP1b
Regulates the positive
regulator of flowering
CONSTANS (CO)
C terminal motif interacts with TOPLESS Co-repressor
Failure to induce FLOWERING LOCUS T
Induced late flowering
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• These occur as a result of processes such as splicing,
alternative translation start and stop site choices, which can
give rise to mRNA isoforms encoding microProteins.
• In addition, microProteins may also be produced by post-
translational processing, such as proteolytic cleavage which
results in smaller products capable of interfering with their
larger, un-cleaved precursor proteins.
Microproteins - Classification
Trans-microProteins cis-MicroProteins
• These are the individual transcription units that are
evolutionarily related to larger genes encoding multi-
domain proteins.
• There is evidence that some microProtein genes evolved
in genome amplification events and subsequent
domain-loss, resulting in single-domain-containing
inhibitory small proteins.
here the system needs to be
activated by certain factors
to form an active complex
the target complex can
be the active complex
until the interaction of
the microProtein with
the target disturbs the
stable target complex
Microproteins - sometimes - Negetive regulators
LITTLE ZIPPERS
Transcriptionally controlled by
HD-ZIP III transcription factor
Negetively regulate the HD-ZIP III transcription
factor proteins with non-productive dimer
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(According to mode of origin)

Plant Microproteins and Light
(Staudt and Wenkel, 2018)
bHLH PIF transcription factors – acting
downstream from the photo receptor system
1
HFR 1 – an atypical bHLH
(trapping PIF factors and -
make non-functional complex)
Suppress excess
shade development
2
HFR 1 also interact with Id
– HLH protein (KDR) and
overexpress it
KDR suppresses
HFR activity
Elongation of
hypocotyls
3
30-07-2020

Plant Microproteins and Hormone Signalling
• The plant Id-like KDR protein sequesters HFR1, shifting
the equilibrium to PIFs and allowing them to form active
homodimers.
• Overexpression of the bHLH target (IBH1) causes
decreased leaf–lamina inclination in rice and dwarfism in
Arabidopsis, and overexpression of the Id-like proteins
PRE or ILI1 increases the leaf–lamina inclination in rice
and induces petiole elongation in Arabidopsis.
bHLH - basic helix–loop–helix
BR - brassinosteroid hormone
HLH - helix–loop–helix
IBH1 - ILI1 binding bHLH
Id - inhibitor of DNA binding
KDR - KIDARI
PIF - phytochrome-interacting factor
30-07-2020

Plant Leucine-Zipper-Like MicroProteins
In wild-type plants, HD-ZIPIII
transcription factors bind to DNA as
homodimers to control developmental
processes including meristem and leaf
development.
If HD-ZIPIII activity is reduced or lost, or if
ZPR proteins are over-expressed, the shoot
meristem can terminate and leaves show
downward curling.
In hd-zip III gain-of-function mutant
plants or multiple zpr loss-of-
function mutant plants, the meristem
becomes larger and disorganized
and leaf development can be
disturbed.
30-07-2020

Other Plant MicroProteins
• KNOTTED-related homeobox (KNOX) domain is conserved in plant KNOX genes.
• Animal myeloid ectopic viral integration site (MEIS) proteins also have a KNOX domain and a
TALE homeodomain, which was subsequently called MEINOX (Bürglin, 1998).
• In Arabidopsis, two classes of KNOX genes exist, each of which includes four genes encoding
MEINOX-TALE-homeodomain proteins, here referred to as KNOX proteins.
• Homeodomain transcription factors belonging to
the three-amino-acid loop-extension (TALE) family
of proteins are also targets of miPs in plants.
• TALE homeodomain transcriptional regulators are
characterized by an insertion of three amino acids
between α-helices 1 and 2 of the homeodomain.
• TALE homeodomain proteins are evolutionarily
conserved and exist in animals, fungi and plants,
indicating that they evolved in a common ancestor.
https://www.researchgate.net/publication/326045715_Prep1_A_Homeodomain_Transcription_Factor_Involved_in_Glucose_and_Lipid_Metabolism/figures?lo=1
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Software Programme (miP3) for Predicting
MicroProteins and Their Target TFs
• The algorithm is called miP prediction program (miP3) which is helpful to determine biological roles and
evolution of miPs.
• Moreover, miP3 can be used to predict other types of miP-like proteins that may have evolved from other
functional classes such as kinases and receptors.
• The program is freely available and can be applied to any sequenced genome
(de Klein et al., 2015)
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Identification of Microproteins Using
Bioinformatics Approaches - miPFinder
• Protein size
• Domain organization
• Known protein interaction
• Evolutional origin of microproteins
Small open-reading frames – Identified by Ribosome sequencing
(Example – CYREN and NoBody)
CAGE – Cap analysis of gene expression –
Identify and quantify transcription start and
termination site using short sequence tag
Process – PROTOMAP, COFRAIDC, ATOMS
and TAILS – uses Mass Spectometry
Yeast 2 Hybrid System
1
2
3
4
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Biotechnological Relevance of MicroProteins
(A New Tools to Control Protein Activity)
• In Arabidopsis, the overexpression of the PPI domain of the transcription factors
SUPPRESSOR OF OVEREXPRESSOR OF CONSTANS 1 (SOC1), AGAMOUS
(AG) and LATE ELONGATED HYPOCOTYL (LHY) resulted in phenotypes similar
to the loss-of-function mutant of the respective transcription factors. (Seo et al., 2012)
• This approach was also effective when the PPI domain of SOC1 was overexpressed in
Brachypodium distachyon resulting in delayed heading. (Eguen et al., 2018)
• Synthetic microProteins have also been used to successfully modulate flowering time
of rice grown in long day conditions. These studies reveal that the ectopic expression
of the PPI domain of transcription factors can function as microProteins due to their
ability to negatively regulate the larger transcription factors.
30-07-2020

Case Study - I
• They have classified all Arabidopsis thaliana microProteins and developed a synthetic
microProtein approach to target specific protein classes, such as hydrolases, receptors and
lyases.
• Their findings reveal that microProteins can be used to influence different physiological
processes, which makes them useful tools for posttranslational regulation in plants.
Background : -
(Dolde et al., 2018)
Plant physiology
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• HYPONASTIC LEAVES1 (HYL1)
• SERRATE (SE)
• DAWDLE (DDL)
• PIWI/ARGONAUTE/ZWILLE (PAZ) domain of DCL1 mediates the interaction with SE
and DDL.
• BRI1 KINASE INHIBITOR1 (BKI1) maintains BRI1 in an inactive form through
heterodimerization at the kinase domain and blocks the interaction with the coreceptor
BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1).
• CRY1 belongs to the flavoprotein photoreceptor family found in plants and animals. In
plants, CRY1 and its homolog, CRY2, mediate blue light-induced responses, such as de-
etiolation and photoperiodic flowering.
• Both CRY1 and CRY2 need to form homodimers to execute their photoreceptor activity.
Components explored in this experiment
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Classification of MicroProtein Candidates
• MicroProtein candidates for Arabidopsis TAIR10 were taken from a published list (Straub and Wenkel, 2017) and only
conserved candidates were selected.
• The PANTHER (Protein ANalysis THrough Evolutionary Relationships; Mi et al., 2017) protein class (Mi et al., 2013) of the
putative ancestor with highest sequence similarity to the microProtein was determined.
• All annotated nontranscription factor-related microProtein candidates were grouped into the 19 remaining parental classes. A
one-sided Fisher’s exact test for overrepresentation was performed with R Version 3.4.1.
Overview of conserved microProtein
candidates in Arabidopsis and the protein
class of their putative ancestors
Fisher’s exact test was used to test for
significant overrepresentation of
microProtein candidates (P , 0.05).
NS - Not significant
To determine whether microProteins can regulate a wide
range of protein classes, they developed a synthetic
microProtein approach. They selected DICER-LIKE1
(DCL1), BRASSINOSTEROID INSENSITIVE1 (BRI1), and
CRYPTOCHROME1 examined if synthetic microProteins
could regulate their activity
Percentage of non-transcription factor-
related microProtein candidates and the
protein class of their putative ancestors.
1. Helicase involved in microRNA (miRNA)
biogenesis
2. Brassinosteroid (BR) signal transduction
3. Lyases - enzymes break chemical
bonds of compounds to generate two products
Findings -
30-07-2020

Alteration of miRNA Biogenesis by miP-DCL1
• Images of two representative independent 35S::miP-DCL1 lines.
Phenotypes were compared with Col-0 and two DCL1 mutants, dcl1-2
and dcl1-11.
• Compared with Col-0 wild-type plants, they showed an overall reduction
in size and resembled dcl1 mutant plants
• Domain architectures of DCL1 and miP-DCL1 - RNA-binding domain.
• RT-qPCR results showing expression of endogenous DCL1 and transgene
miP-DCL1 in 35S::miP-DCL1 lines compared with Col-0.
• Experimental replicates were statistically tested using a Student’s t test (*P ,
0.05) and the ability of the PAZ domain to homodimerize was tested using a
yeast two-hybrid assay.
• Normal yeast growth was observed for the serial dilutions on nonselective
SD medium lacking Leu and Trp (SD-L-W) and only positive interactors
grew on selective SD medium lacking Leu, Trp, and His (SD-L-W-H)
supplemented with 2.5 mM 3-aminotriazole (3-AT).
• To generate transgenic plant lines overexpressing the synthetic microProteins, the cDNAs, encoding the protein-protein interaction
domains, were cloned into the binary vector pJAN33 containing a CaMV35S promoter, a FLAG tag, and a BASTA (glufosinate)-
resistance (BAR) gene. Constructs were then transformed into Arabidopsis via floral dip methode.
• RT-qPCR was performed using a KAPA SYBR FAST qPCR Kit (Kapa Biosystems) on a Bio- Rad CFX384 machine.
30-07-2020

Alteration of miRNA Biogenesis by miP-DCL1
• Small RNA blot analyses display the levels of miRNAs in
35S::miP-DCL1 lines compared with Col-0 and se-1 using
5.8S rRNA as a loading control.
• DCL1 forms a complex with HYL1, SE, and DDL to process
miRNA.
• The complex may be regulated by an unknown suppressor
protein.
• MiP-DCL1 enhances the DCL1 complex function by
sequestering the complex inhibitor in a nonfunctional dimer.
A model depicting miP-DCL1 function
Continued ..
Small RNA Isolation and Northern Blot Analysis - Total RNAs were isolated from 14-d-old seedlings using Trizol
reagent (Invitrogen). The extracted aqueous phase was precipitated with 2 propanol twice (100% and 75%) and
resolved in 50% formamide. Purified RNAs were resolved on a 5% to 15% gradient denaturing polyacrylamide gel
(National Diagnostics) before transfer to a nylon membrane (Amersham).
Findings -
30-07-2020

BRI1 Activity Can Be Controlled through the
Transmembrane-Juxtamembrane Domain of BRI1
• The BRI1 homodimer is inactivated by BKI1.
• BL binding activates BRI1 and causes dissociation of BKI1 and association of BAK1.
• The synthetic microProtein miP-BRI1 prevents BRI1 activation during BL binding.
Model of BRI1 activation and miP-BRI1 regulation
• Yeast Two-Hybrid Assays (Protein interaction assays) were performed using the Matchmaker yeast twohybrid system (Clontech).
Col-0, bri1-5, det2, and two 35S::miP-BRI1 lines treated with mock
(2BL) or 100 nM BL (+BL)
30-07-2020

• RT-qPCR showing expression of endogenous
BRI1 and transgene miP-BRI1 in 35S::miP-
BRI1 lines compared with Col-0.
• Experimental replicates were statistically
tested using a Student’s t test (**P,0.01).D,
Altered CPD expression in Col-0, det2, bri1-
5, and the two 35S::miP-BRI1 lines in
response to 100 nM exogenous BL.
BRI1 Activity Can Be Controlled through the
Transmembrane-Juxtamembrane Domain of BRI1
Continued ..
• miP-BRI1 functions as a synthetic microProtein. Plants ectopically expressing miPBRI1
show a mild dwarf phenotype and alteration in the expression of CPD in response to BL
application.
• Thus, miP-BRI1 influences BRI1-mediated signal transduction.
Findings -
30-07-2020

The N-Terminal Photolyase Subdomain of CRY1
Can Inhibit CRY1 Function
• Model depicting CRY1 function and miP-CRY1 inhibition. The CRY1 homodimer is
activated by a blue light signal. Activated CRY1 regulates de-etiolation of the plant.
• MiP-CRY1 forms a non-functional heterodimer with CRY1 that leads to hypocotyl
elongation under blue light.
Domain structures of CRY1 and miP-CRY1
30-07-2020

• Image of representative Col-0, cry1cry2, and two
independent 35S::miP-CRY1 lines grown under white light,
dark, or blue light conditions.
• RT-qPCR showing expression of endogenous CRY1 and
transgene miP-CRY1 in 35S::miP-CRY1 lines compared
with Col-0.
• Experimental replicates were statistically tested using a
Student’s t test (**P , 0.01).
• Interaction of miP-CRY1 with CRY1 was tested using a
yeast two-hybrid assay.
• Normal yeast growth was observed for the serial dilutions
on nonselective medium lacking Leu and Trp (SD-L-W).
• Only positive interactors grew on selective medium lacking
Leu, Trp, and His (SD-L-W-H) supplemented with 10 mM
3-aminotriazole (3-AT).
The N-Terminal Photolyase Subdomain of CRY1
Can Inhibit CRY1 Function
Continued ..
• These results revealed that plants overexpressing miP-CRY1 behave like cry1cry2 double mutant plants,
and miP-CRY1 is able to interact with the full-length CRY1 protein.
• They also demonstrated that the PH domain of CRY1 can be used to negatively regulate CRY1 protein
activity in a microProtein-type manner and thus influence blue light responses of the plant.
Findings -
30-07-2020

Case Study - II
(Eguen et al., 2020)
Journal of Integrative Plant Biology
• In Arabidopsis thaliana, the B‐box zinc finger transcription factor CONSTANS (CO) positively regulates the florigen gene
FLOWERING LOCUS T (FT) to activate flowering in long days.
• Flowering in rice is modulated by the CO homolog HEADING DATE 1 (Hd1), which regulates the expression of the
florigens HEADING DATE 3A (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) in short day (SD) and long day
conditions (LD)
• B‐box proteins are a conserved group of proteins that are involved in growth and development. In Arabidopsis, two B‐box
containing microProteins, microProtein1a (miP1a) and microProtein1b (miP1b), are also involved in the regulation of CO
to control flowering time.
• To explore the possibility of using synthetic microProteins to change the cycle length of cereal crops, they created a
synthetic Hd1 microProtein to interfere with the endogenous function of CO homolog, Hd1, in rice.
• To achieve this, they used the B‐Box domains of Hd1 and expressed it as an individual FLAG‐tagged fusion protein in
transgenic rice plants.
Background : -
30-07-2020

Protein Expression Profile of Hd1 and Hd1miP
• The Hd1 and Hd1miP forms were overexpressed in WT Nipponbare plants. The protein expression
of OX‐Hd1miP and OX‐Hd1 was measured and the three overexpressors of Hd1miP and the highest
overexpressor of OX‐Hd1 were selected for further experiments
• To determine if a synthetic Hd1 microProtein (Hd1miP) and Hd1 protein can interact, their
interaction was tested in yeast. Hd1miP fused to the GAL4 binding domain (BD) and Hd1 fused to
the GAL4 activation domain (AD) were transformed into yeast.
• Yeast growth was observed on media lacking leucine, histidine, and tryptophan (‐L/‐H/‐W) in the
presence of 10 mM 3‐aminotriazole, which is evidential of Hd1 and Hd1miP interaction.
Schematic diagram of Hd1 and Hd1miP
loading control
30-07-2020

Effect of The Overexpression Hd1miP on Flowering
• The heading dates of WT, OXHd1miPs, OX‐Hd1, and hd1‐1 plants were determined in short day (SD) (10 h light/14 h dark), long day
(LD) (14.5 h light/9.5 h dark), and strong long day (SLD ‐ 16.5 h light/7.5 h dark) conditions.
• In SD, no significant difference was observed between the lines. In LD, OX‐Hd1miP‐1 and OX‐Hd1miP‐2 showed 11% and 17%
earlier heading dates than WT; hd1‐1 showed 34% earlier heading date. OX‐Hd1miP‐W plants, which had the lowest Hd1miP protein
expression, showed late heading dates similar to WT.
• In SLD, OX‐Hd1miP‐1, OX‐Hd1miP‐2, and hd1‐1 plants showed 34%, 32%, and 49% earlier heading dates than WT respectively;
OX‐Hd1miP‐W plants showed late heading dates similar to WT and OX‐Hd1 showed 34% later heading dates than WT.
• Taken together, the results show that the overexpression of Hd1miP results in early flowering in LD and SLD and the effect on
flowering time is dependent on the abundance of Hd1miP protein.
• The flowering phenotypes of OX‐Hd1miP‐1 and OXHd1miP‐ 2 reveal a microProtein type inhibitory mechanism by which Hd1miP
can inhibit the repressor activity of Hd1 in LD thereby giving rise to earlier flowering plants.
30-07-2020

Effects of The Overexpression of Hd1miP on
The Endogenous Levels of Hd1
• The expression level of Hd1 was quantified by qPCR using primers that
recognize the 3′ untranslated region (3′ UTR) of Hd1.
• The Hd1 levels in OX‐Hd1miP‐1 and OX‐Hd1miP‐W were similar to WT
suggesting that the presence of the synthetic Hd1miP does not affect
endogenous Hd1 levels.
• Conversely, OX‐Hd1miP‐2 lines showed endogenous Hd1 levels that were
much lower than in WT, suggesting a possible suppression of Hd1
expression in OX‐Hd1miP‐2.
• No expression of Hd1 was detected in hd1‐1 plants.
• Attempts to correlate Hd1 gene expression in the different OX‐Hd1miPs
lines with their corresponding flowering phenotype revealed that the
regulation of Hd1 expression levels is not the determining factor in the
early flowering phenotype of OX‐Hd1miPs.
• To analyze the expression of the B‐box region of Hd1, Hd1 B‐Box primers
were used to quantify Hd1 levels. The levels of Hd1 were higher in all
Hd1miP overexpressor lines than in WT and no Hd1 expression was
observed in hd1 mutant plants.
30-07-2020

Expression Profile of Florigens Hd3a and RFT1
• The expression profile of florigens Hd3a and RFT1 was analyzed in the different lines to obtain additional
information on the molecular mechanisms underlying the flowering time phenotype.
• The hd1‐1 plants showed higher levels of Hd3a than WT due to the absence of the Hd1 repression. In
OX‐Hd1miP plants, the levels of Hd3a were similar to WT. The expression of RFT1 was higher in all Hd1miP
overexpressors than in WT and hd1‐1.
• This indicates that although OX‐Hd1miP and hd1‐1 result in early flowering in LD, they differ in the regulation
of Hd3a and RFT1. So, Hd1 suppresses the expression of the florigens Hd3a and RFT1 in LD plants.
30-07-2020

Test of Viability and Productivity of
OX‐Hd1miP Plants
• To determine the viability and productivity of OX‐Hd1miP plants in LD, the panicle characteristics were
measured 35 d after the first panicle emergence.
• The grain width and grain length of OX‐Hd1miPs was similar to WT with the exception of OX‐Hd1miP‐1,
which showed slightly longer grains and hd1‐1 plants which showed slightly wider grains.
• Measurements of the moisture content revealed hd1‐1 plants to have 160% increased moisture content,
OX‐Hd1miP1 and OX‐Hd1miP‐2 had 56% increased moisture content, while OX‐Hd1miP‐W showed
similar moisture content to WT.
• This suggests that Hd1 may also be involved in the control of the moisture content in rice grains but it does
not have a strong effect on grain size.
30-07-2020

Hd1 Levels and Activity Affects Maturity Rate of The Panicles
• In hd1‐1 plants, 39% of the panicles were mature while 38% and 36% were mature in OX‐Hd1miP‐1 and OX‐Hd1miP‐2,
respectively. This suggests that Hd1 levels and activity may also play a role in the maturity rate of the panicles.
• The OX‐Hd1miP‐1 and OX‐Hd1miP‐2 plants showed an average spikelet fertility of 31% and 36%, which is less than the
63% spikelet fertility in the WT; hd1‐1 plants had an average spikelet fertility of 37%.
• When the spikelet fertility of only mature panicles was calculated there was an approximately 100% increase in spikelet
fertility in both the OX‐Hd1miPs and hd1‐1 lines compared to the 40% increase in WT.
• The average total number of grains produced in the OX‐Hd1miP and hd1‐1 lines was also less than WT plants. Hd1 seems
to also affect the fertility rate and the number of grains produced irrespective of Hd1 being inhibited at a transcriptional or
at a post‐translational level. Extending the harvest time beyond 35 d may significantly increase the yield of OX‐Hd1miP
and hd1‐1 plants.
30-07-2020

Hypothetical Model of Synthetic
MicroProteins Affect Hd1 Protein Activity
• Flowering is promoted by Hd3a and RFT1. Ehd1 induces RFT1 regardless of photoperiod and
enhances Hd3a in response to short photoperiod.
• Hd1 acts as a repressor of both Ehd1 and Hd3a when exposed to long photoperiods. In response
to short photoperiods Hd1 activates Hd3a.
• In the presence of the synthetic microProtein (Hd1miP), Hd3a expression is increased regardless
of the photoperiod which results in early flowering in both short day (SD) and long day (LD)
conditions. They also observed increased expression of RFT1 which could be an indirect effect.
30-07-2020

Summary
• Dolde et al., 2018 classified all Arabidopsis thaliana microProteins and developed a
synthetic microProtein approach to target specific protein classes, such as hydrolases,
receptors, and lyases, in a proof-of-concept approach which revealed that microProteins
can be used to influence different physiological processes, which makes them useful
tools for posttranslational regulation in plants and potentially also in animals.
• The recent findings of Eguen et al., 2020, demonstrated how a synthetic microProtein‐
based system is effective at changing flowering time in rice. The inhibition of the long
day repression mechanism of rice might have beneficial implications in the expansion of
rice cultivation in equatorial regions and areas of northern latitudes.
• Synthetic microProteins are proving to be a useful tool in targeting and inhibiting
proteins in a more specific manner with the reduced likelihood of an off‐target effect
(Bhati et al. 2018; Dolde et al. 2018).
• Furthermore, synthetic microProteins can also aid in deciphering what processes are
dependent on the activity of the inhibited target protein. However, using microProteins
as a tool involves knowing the interaction domain of the protein of interest and the
ability of the microProteins to form heterodimers with the target.
30-07-2020

References
1. Bhati, K. K., Blaakmeer, A., Paredes, E. B., Dolde, U., Eguen, T., Hong, S. Y., & Wenkel, S. (2018). Approaches to
identify and characterize microProteins and their potential uses in biotechnology. Cellular and Molecular Life
Sciences, 75(14), 2529-2536.
2. de Klein, N., Magnani, E., Banf, M., & Rhee, S. Y. (2015). microProtein Prediction Program (miP3): a software for
predicting microProteins and their target transcription factors. International journal of genomics, 2015.
3. Dolde, U., Rodrigues, V., Straub, D., Bhati, K. K., Choi, S., Yang, S. W., & Wenkel, S. (2018). Synthetic
microproteins: versatile tools for posttranslational regulation of target proteins. Plant physiology, 176(4), 3136-3145.
4. Eguen, T., Ariza, J. G., Brambilla, V., Sun, B., Bhati, K. K., Fornara, F., & Wenkel, S. (2020). Control of flowering in
rice through synthetic microProteins. Journal of Integrative Plant Biology, 62(6), 730-736.
5. Staudt, A. C., & Wenkel, S. (2011). Regulation of protein function by ‘microProteins’. EMBO reports, 12(1), 35-42.
6. Sahoo, J.P., Behera, L., Sharma, S.S., Praveena, J., Nayak, S.K. and Samal, K.C. (2020) Omics Studies and Systems
Biology Perspective towards Abiotic Stress Response in Plants. American Journal of Plant Sciences, 11, 2172-2194.
https://doi.org/10.4236/ajps.2020.1112152
https://www.researchgate.net/profile/Kaushal_Bhati/publications
30-07-2020

30-07-2020

SYNTHETIC MICRO PROTEINS - VERSATILE TOOLS FOR THE REGULATION OF PROTEIN FUNCTIONS IN CROP PLANTS

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