agriculture

1. Presenter:
Ahmed Wessam Ahmed Abd Already
(Ph.D. candidate)
Supervisor: Prof. Imran Haider Shamsi
Department of Agronomy, College of Agriculture and Biotechnology,
Zhejiang Univ
2023.05.15
Calcium Phosphate Nanoparticles and Piriformospora indica
alleviate salinity stress in barley growth in the context of salinity
stress tolerance and their Mechanisms.

2.  Barley (Hordeum vulgare L.)
 Barley (Hordeum vulgare L.) is one of the most important crops globally, with multiple uses (Han et al.,
2020).
 Moreover, it is also an ideal crop for producing healthy food as barley grains contain high content of β-
glucan, flavonoid, and bioactive compounds. Meanwhile, barley is considered as a classical model plant
for physiological and genetic studies on cereal plants (Gürel et al., 2016).
Research background (Introduction):

3.  The root endophytic basidiomycete P. indica belongs to the order Sebacinales.
 P. indica forms a classical type of mutualistic mycorrhizal symbiosis with a broad spectrum
of host plants, including barley, Arabidopsis, wheat, maize, and tomato (Ghaffari et al., 2019).
 P. indica shows various functions, such as promoting plant growth, enhancing stress
tolerance and disease resistance, and accelerating nutrient acquisition and bioactive substance
accumulation (Yang et al., 2020). The biochemistry data indicated that intracellular Ca2+
concentrations were increased in response to the early stage of root endophyte colonization
.In addition, molecular research showed that genes involved in hormone signaling, cell wall
metabolism, transporting carbohydrate metabolism, and root formation were upregulated after
P. indica colonization (Lee et al., 2011).
 Unfortunately, the biological functions and potential regulatory mechanisms of barley
lncRNAs in response to the colonization of P. indica remain unclear and require further
investigation.
 Piriformospora indica (P. indica)

5.  Recently, calcium phosphate
nanoparticles (CaP NPs) have gained great
attention worldwide because of their
promising agricultural applications.
 The application of CaP NPs to the plant
system has proven to be efficient in
promoting plant growth. For instance,
Rane et al. (2015) reported that the
application of CaPO4 NPs demonstrated
root proliferation, harmonious growth
elevation, and vitality development
possessions along with an improvement in
endosymbiotic and arbuscular mycorrhizal
fungi in maize.
 Calcium Phosphate Nanoparticles
(CaP NPs) :

6.  In another study conducted on rice, it was concluded that CaPO4 modifies growth as well as
antioxidant rejoinders in a dose-reliant way. It was found that CaP NPs interacted with plants
and inflicted physiological changes (Upadhyaya et al., 2017).
 Corresponding to their structure and capability to incorporate other chemicals, CaP NPs are
also being doped with other nutrients for their maximum utilization in precision agriculture.
 Recently, nano calcium phosphate (NCaP) were tested on the production of Snap Bean
(Phaseolus vulgaris L.) plants, and it was found that these meaningfully augmented the shoot
as well as root biomass, nutrient composition of shoot and root, the yield constituents, the
nutrient proportion, and crude protein fraction in pods of the snap bean plants as equated to
the conventional P supplementation (El-Ghany et al., 2021).The roles of CaP NPs in
alleviating salinity stress induced crop toxicity are still elusive.
 Calcium Phosphate Nanoparticles (CaP NPs) :

7.  Although it was recently determined that CaP-NPs and P.indica can alleviate salinity stress in
barley, the effects of CaP-NPs and P.indica properties and the molecular mechanisms are still
unclear.
 The mechanisms CaP-NPs and P.indica mediated plant growth strengthening are still unclear.
 Due to the variable environments in the field and its complex quantitative character, little
progress has been made on improvement of barley varieties for salt tolerance. Limited genetic
studies of barley salt tolerance have been carried out until now due to the lack of an efficient
index for measuring salt tolerance.
Problem (Study significance):

8.  The objectives of the current study will determine the beneficial effects of CaP-NPs and
P.indica on growth and physiological traits of barley genotypes differing in salt tolerance
under salinity stress. In addition, the roles of CaP-NPs and P.indica in reducing Na+
bioavailability and acro-petal translocation through its regulation of genes will also examine ,
to determine if CaP NPs and P.indica can be used as a nano-fertilizer and bio-fertilizer to
alleviate Na+ toxicity and accumulation in barley. And we evaluated the effect of CaP-NPs
and P.indica on the growth and sugar and hormone metabolisms of barley.
 To study, we will use the non-invasive microelectrode ion flux estimation (MIFE) technique
to investigate the mechanistic basis of Ca2+, Na+, H+ and K+ amelioration of the salinity stress
in barley (Hordeum vulgare). By using a range of microelectrode and imaging techniques, we
will show that the beneficial effects of CaP-NPs and P.indica on the salt tolerance.
Developing of suitable methodology to investigate the effect of salinity stress on barley.
Research objectives:

9.  Genotypes will be used to measure Ca2+, Na+ and K+ retention in cytosol. And measure
reactive oxygen species related experiments
 To gain insights into the molecular mechanisms underlying the CaP-NPs and Piriformospora
indica -mediated alleviation of salinity stress in barley. And to identify genes and regulatory
pathways that are modulated by CaP-NPs and Piriformospora indica in barley plants, leading
to salt tolerance.
Research objectives:

10.  Due to the variable environments in the field and its complex quantitative character, little
progress has been made on improvement of barley varieties for salinity tolerance. Limited
genetic studies of barley salinity tolerance have been carried out until now due to the lack of
an efficient index for measuring salinity tolerance.
 Surprisingly, to the best of our knowledge not a single breeding work has attempted to link
salinity stress and ROS stress tolerance using QTL approach. Na+, K+ in cytosol, membrane
potential and ROS will be linked, and this gap of knowledge will be overcome in this study.
 Although it was recently determined that CaP-NPs and P.indica can alleviate salinity stress in
barley, the effects of CaP-NPs and P.indica properties and the molecular mechanisms are still
unclear.
 The mechanisms of CaP-NPs and P.indica mediated plant growth strengthening are still
unclear, especially under hydroponic experiment .
Significance of study:

11. This study will be divided into four stages.
1. Effect of Calcium Phosphate nanoparticles and Piriformospora indica on physiological and
biochemical in barley in the context alleviation of salinity stress tolerance.
2. Effect of Calcium Phosphate nanoparticles and Piriformospora indica on physiological and
molecular mechanisms mediating xylem Na+ loading in barley in the context alleviation of
salinity stress tolerance.
3. Electrophysiological for ion flux measurements using the MIFE technique.
4. Total RNA isolation, cDNA preparation, and gene expression .
The main research contents and plans to solve the key technology:

12. The experiments will be carried out in the Hydroponic culture and lab
(MIFE experiments) at Zhejiang University. four varieties of barley will
be used in both experiments to make and validate the methodology. Two
treatments will be used for the whole experiment one is calcium
Phosphate nanoparticles and Piriformospora indica under salinity stress.
.
The research plan and technical route

13.  Experiment will unleash the physiological and biochemical changes and mechanism
that occurred during the synergism of CaPNPs and P. indica for alleviating the
salinity stress in barley.
 Treatments: 07.
 TrT0: Control (CK)
 TrT1: CaP NPs.
 TrT2: P.indica.
 TrT3: NaCl .
 TrT4: CaP NPs + P.indica.
 TrT5: CaP NPs + NaCl.
 TrT6: P.indica + NaCl.
 TrT7: CaP NPs + P.indica + NaCl
 Varieties of barley: Two varieties will be
salinity sensitive and two will be salinity
tolerant.
 Levels/Replicates: 24
 Concentration:
o CaP NPs: ...…… mM/l.
o NaCl: 0, 50, 100, 200, and 300 μM/l.
Experimental design:

14. Plant material
 Reactive Oxygen
Species,
Malondialdehyde, and
Histochemical
Analysis.
 Antioxidant
Enzymatic Activities.
 Determination of
endogenous hormones.
 Determination of
soluble sugar.
 Secondary
metabolites.
P.indica -inoculation
Barley genotypes (Hordeum vulgare L.)
(Hordeum vulgare L.)
Molecular
studies
Control
 Gene
expression
via qRT-
PCR.
 Transcriptom
ic profiling
of roots or
shoots.
 Metabolomic
s study.
 Characterization of
calcium Phosphate
Nanoparticles.
 Plant sampling,
nutrient profiling, and
metal analysis.
 Determination of net
photosynthetic rate and
chlorophyll content.
 Root Na+ content
determination
 Leaf sap Na+ and K+
content.
 Xylem Na+ and K+
content.
NaCl CaP NPs
Biochemical
analyses
Treatments
Non-inoculation
Physiological studies
Measurements
Data collection, statistical analysis, and results
interpretation
Electrophysiological
studies
 Na+, K+, Ca2+ and
H+ flux
measurements using
MIFE.
 The transmembrane
electrical potential
difference (Em)
measurements.
NaCl + CaP NPs

15.  Before use, CaP NPs will sonicate for 1 h in each suspension. Three independent replicates
will set for each treatment and each replicate contained twenty barley seedlings. The
hydroponic experiment will arrange with randomized design in a glasshouse, with
photoperiod of 16/8h light/dark, 22 /18◦C temperatures, light intensity of 225 ±25 µmol m-2
s-1 and around 60 % of relative humidity.

16. Physiological studies and Biochemical analyses.
1- Characterization of nanoparticles.
Briefly, we will perform SEM, TEM, and FTIR spectral analyses of Calcium Phosphate
Nanoparticles (CaP NPs).
2- Plant sampling, nutrient profiling, and metal analysis.
Briefly, Effect of exogenous CaP-NPs application and P.indica on growth of barley genotypes
under salinity stress on plant high (cm), root length (cm), shoot DW (g plant -1); and root DW
(g plant -1). Then will compare with the control.
3- Root Na+ content determination.
Briefly, Effect of exogenous CaP-NPs application and P.indica on microelement
concentrations in roots of barley genotypes under salinity stress. Then will compare with the
control.
Parameters to be measured:

17. 4- Xylem and leaf sap Na+ and K+ content.
Briefly, to further explore the subcellular distributions of Na+ and K+, the total Na+ and K+
contents of the in Xylem and s Leaves will determine under salinity stress and effect of
exogenous CaP-NPs application and P.indica. Then will compare with the control.
5- Determination of net photosynthetic rate and chlorophyll content.
Briefly, Measure effects of Cao NPs and P.indica on SPAD value (arb. Units), Fv/m value (arb.
Units), net photosynthetic rate, and transpiration rate under salinity stress. Then will compare
with the control.
6- Reactive Oxygen Species, Malondialdehyde, and Histochemical Analysis and antioxidant
Enzymatic Activities.
Briefly, Effect of exogenous CaP-NPs application and P.indica on antioxidative capacity in
shoots and roots of barley genotypes. MDA and H2O2 contents in shoots and roots; SOD, CAT
and APX activities in leaves and roots; ASA, GSH and GR contents in shoots and roots of barley
seedlings. Then will compare with the control.
Parameters to be measured:

18. 7- Determination of endogenous hormones.
Briefly, Measure Effect of exogenous CaP-NPs application and P.indica on the contents of CTK,
GA3, IAA and ABA in leaf sample and stem sample and compared with the control
8- Determination of soluble sugar.
Briefly, Effect of exogenous CaP-NPs application and P.indica on the contents of glucose,
sucrose, and fructose in barley leaf sample and stem sample.
Parameters to be measured:

19. Electrophysiological studies: MIFE non-invasive ion flux measurements.
 Na+, K+, Ca2+ and H+ flux.
To study the kinetics of the ion flux of Na+, K+, Ca2+ and H+ flux using MIFE under P. indica colonization in
barley.
 The transmembrane electrical potential difference (Em) measurements.
Before measurements, roots of an intact 7-day-old barley seedling will be immobilized in BSM for 30 min.
The Em measurements will perform as describe by Gill et al. (2018). Conventional 1 M KCl filled Ag-AgCl
microelectrodes will connect to the MIFE electrometer via an Ag/AgCl half-cell. During the Em
measurement, the microelectrode will be impaled into the external cortex cells of the mature zone (⁓6 mm
the from root tip) with a manually operate 3D micromanipulator (MHW-4, Narishige, Tokyo, Japan). Once a
stable Em measurement will obtain for 1 min, combined low-pH/ AlCl3 will be initiated. The resulting
change in the transient Em will continuously be monitored for up to 25:30 min. Em values will record by the
MIFE CHART software (Shabala, 2006). For each treatment, Em values of 5–6 individual seedlings will
average
Parameters to be measured:

20.  Molecular studies: Total RNA isolation, cDNA
preparation, and gene expression.
Briefly, Effects of CaP-NPs and P.indica application on expression of 30 genes in shoots and
roots of barley genotypes after 20 days of NaCl exposure. Then will compare with the
control.
o Gene expression via qRT-PCR.
o Transcriptomic profiling of roots or shoots.
o Metabolomics study.

21. The existing research condition and research schedule
Experiment Status
Expected time to
finish
1 In progress -
2 In progress -
3 In progress -
4 In progress -

22.  CaPNPs and P. indica alleviate salinity stress in barley.
 Exogenous application of CaPNPs and P. indica in barley promote the growth and improve the
physiological indices of barley CaPNPs and P. indica treatment also improves barley
photosynthesis, promotes sugar synthesis and transport, and increase the endogenous
hormone level in barley CaPNPs and P. indica will be use as supplements for chemical
fertilizers to improve barley performance in the field.
These outcomes are expected at the end of the
study:
The expected results, the innovation of this study :

23.  A suitable methodology can be suggested to new researchers working related to salinity stress
at the end of the study. And a few reports have demonstrated the genetic variation of the
tolerance to salinity stress in cereal crops. This gap of our knowledge will be overcome.
 The study employs an integrated approach by combining metabolic, transcriptomic, MIFE
(Microelectrode Ion Flux Estimation), and gene expression techniques. This multidisciplinary
approach allows for a comprehensive analysis of the interactions between calcium phosphate
nanoparticles (CaP NPs), P .indica, and salinity stress in barley.
 The study aims to provide novel insights into the mechanisms underlying the alleviation of
salinity stress in barley. By integrating various techniques, it can reveal new pathways, genes,
and metabolic processes involved in this interaction. The use of MIFE techniques adds a
unique dimension to the study. It enables the evaluation of the activity of membrane ion
channels, specifically sodium transporters. This innovation provides direct measurements of
ion fluxes and contributes to a better understanding of the physiological responses of barley
plants to CaP NPs and P.indica.
The innovation of this study:

24.  The study incorporates a metabolomics approach to investigate the metabolic changes induced by
CaPNPs and Piriformospora indica in barley plants under salinity stress. This innovation allows for a
holistic analysis of the metabolic pathways involved and their modulation in response to the treatments.
By integrating multiple omics approaches, the study aims to achieve a systems-level understanding of
the interaction between CaPNPs , Piriformospora indica, and salinity stress in barley. This holistic
perspective can reveal complex regulatory networks and interactions, providing valuable information
for future research and potential applications in agriculture.
 Surprisingly, to the best of our knowledge not a single breeding work has attempted to link salinity
stress and ROS stress tolerance using QTL approach. This major gap in our knowledge will be
overcome in this study.
 K+ retention and membrane potential experiments will be used to construct QTL maps. In this project,
the positions of genes conferring cytosolic Na+, K+ retention in salinity stress barley roots will be
mapped.
The innovation of this study:

25. Thank
you