anaymalpani

1. Research strategies on investigating the genetic control
of nutrient transport and distribution in rice grain
Dr. Deepak M. Kasote
Presentation
for
“Postdoctoral Fellow (Grain Quality and Nutrition/ GRS Level 1)”
at
The Centre of Excellence in Rice Value Addition (CERVA), IRRI, Varanasi,
India

2. About Me……..
Academic credential – M.Sc. (Biochemistry), Ph.D. (Chemistry)
Post-Doctoral Experience – National & International Institutes
Texas A&M University, USA
The Rowlett Institute,
University of Aberdeen, UK
Yeungnam University,
South Korea
LC-MS and
Metabolomics
Tshwane University
of Technology, South Africa
Agharkar Research
Institute, Pune
IRSHA, BVDU
Pune
(Plant science, pathology,
nanoscience, and encapsulation
for gut health )
(Natural product, Metabolomics
and bioprospecting)
(Natural product metabolism
and human health)
(Food science, nutraceuticals
and gut health)
(Plant physiology
and metabolomics)
(Chemo-profiling, chemometrics,
and bioprospecting)

3. Rice: food and nutritional security
Rice
Grain quality
Food Security Nutritional security
Undernutrition Over nutrition
(2 billion) (2 billion)
• Rice (Oryza sativa) is one of the most important cereal
grains
• Serves as a staple food source for more than half of the
world’s population, and fulfill over 21% of the calorie
intake needs of the world’s population
• Rice grain quality (nutrient quality) have significant role
in global food and nutritional security
• IRRI developed new varieties to tackle
 Food Security : High yield and disease resistant
rice varieties (IR8, IR36 and R64)
 Nutritional security (micronutrient deficiency and over
nutrition) : Fe, Zn, and pro-vit. A dense and Low glycemic rice
varieties (BR29, IR68144, dhan 62 and Swarna)
"Rice Science For A Better World"

4. Rice: food and nutritional securityRice grain: quest of nutrient quality
• Reported varieties of rice: over 40,000 varieties
• Oryza sativa (Asian rice) has two major subspecies:
1. The sticky, short-grained japonica or sinica
2. The nonsticky, long-grained indica rice
• On the basis of processing: 2 type
1. Brown rice (whole grain) - only the inedible hull removed
2. White rice (milled) - bran and germ removed
• During the milling process (brown to white)-nutrients destroyed or
removed
 67% of the vitamin B3
 80% of the vitamin B1
 90% of the vitamin B6
 half of the manganese and phosphorus
 60% of the iron
 all of the dietary fiber, essential fatty acid including essential amino acid
Mackill, D. J., & Khush, G. S. (2018). Rice, 11(1), 18.
(Germ)

5. Rice grain: quest of nutrient quality
• Polished white rice is the most widely consumed form, and unmilled brown rice
is regularly eaten in some cultures
Enrichment/Biofortification of Nutrients in Polished White Rice Endosperm
Staple food Functional food
Inorganic Nutrients Organic Nutrients Biomolecular Nutrients
• Iron
• Zinc
• Magnesium
• Iodine
• Selenium
• Essential amino acids
• Essential fatty acids
• Low glycemic/Resistant
starches
• Lysophospholipids
• Anthocyanins/polypehnols
• Vitamin B complex
• Provitamin A
• Ascorbic acid

6. Strategies of Nutrients Enrichment in Crops
Genomics-assisted
plant improvement
Conventional
plant breeding
Application of
fertilizers
Common strategies in crop nutrient improvement
• Transgenesis - When trait of
interest absent
• Marker Assisted Selection
(MAS) – When trait of interest
is present
• Genomics-assisted plant improvement is rapid, precise and sustainable approach of nutrient loading in
edible portions of seeds
• For this, fundamental understanding about nutrient absorption (or synthetic pathways), transport,
distribution and loading is essential
• Gene discovery and functional genomics integrated with metabolomics will provide the necessary
understanding to develop and evaluate different approaches to manipulate nutrients composition in edible
portions of seeds

7. Current understanding micronutrient absorption, transport, distribution and
loading in rice
• Micronutrient absorption in Rice (Chelation Stratagy)
 Plants cells utilize two separate mechanisms for acquiring
metals
1. Reduction based strategy- Dicotyledonous and non-
graminaceous plants; higher oxidation states of metal is
reduced to lower oxidation state before being transported into
the cell by its specific transporter
2. Chelation Stratagy- Operates mainly in grasses such as rice,
corn and wheat; plants cells release phytosiderophores (PS)
which belong to muginic acid (MA) family and are derived from
the precursor nicotinamine. These molecules bind to metals
and chelate them and specific plasma membrane transporter
proteins import these metal-PS complexes into the plants
Guerinot, Mary Lou. (2007). Proceedings of the
National Academy of Sciences 104, 7311-7312.

8. Current understanding micronutrient absorption, transport, distribution and
loading in rice
• Micronutrient distribution in Rice ( takes place at node)
 Mineral elements, including both essential and toxic elements, are
delivered to different tissues after they are taken up from the roots, but the
mechanism (or mechanisms) underlying the distribution remains poorly
understood
 In graminaceous plants (e.g. rice), the node (the place on a plant stem
where a leaf is attached) is a structurally pivotal organ and a hub for
distributing essential mineral elements taken up by the roots to different
organs
 A rice culm has 13–16 nodes, with only the upper 4 or 5 separated by long
internodes
Yamaji, N., & Ma, J. F. (2014). Trends in Plant Science, 19(9), 556-563.

9. Current understanding micronutrient absorption, transport, distribution and
loading in rice
 Recently, several transporters have been identified in the nodes that provides new insights about the molecular
mechanisms of intervascular transfer of mineral elements in rice. However, most of the transporters localized in the
nodes have not been identified
 The distribution modes for each element is differ in terms of the requirement of specific organs and the
developmental and environmental changes involved, but it remains to be elucidated how mineral elements in the
nodes are distributed
 Interestingly, some transporters, such as iron transporter (IRT1) and boron transporter (BOR1), have been shown to
be regulated at the transcriptional level or/and post-translational level
 Whereas, some other transporters for mineral uptake are consistently expressed. For example, a major rice
transporter (OsNramp5) for manganese (Mn) uptake does not respond to external low and high Mn concentrations
• Micronutrient distribution in Rice ( takes place at node)

10. Current understanding micronutrient absorption, transport, distribution and
loading in rice
• Micronutrient Loading in Rice Endosperm
 In Rice, large amounts of grain micronutrients may remain in the outer aleurone layers
of the grain. This mainly because, micronutrients loaded into seeds arrives either via
xylem vessels or via the sieve tubes of the phloem, and both paths circulate around
the seed coat.
 Nutrients are not directly unloaded into the endosperm, However, exact reasons why
they are not loaded into the endosperm are not well understood
 However, most importantly, there is evidence that genotypic variation in nutrient
loading in rice endosperm
 Several barriers such as phytic acid that acts as inhibitors for endosperm
internalization of micronutrients
Sperotto, R. A. (2013). Frontiers in plant science, 4, 464.

11. Hypothesis and Central Goal of Proposed Study
“ To investigate the genetic basis of nutrients transport, and distribution
and loading in the rice endosperm using integrated genomic-metallomic
approach”
Hypothesis
“Screening of rice genotype(s) with high seed micronutrients content will
provide unique gene pools for genomics-assisted crop improvement”
Central Goal

12. Objectives of Proposed Study
1. To investigate genotype based variation in polyphenols, vitamins, essential amino
acids, fatty acids and amylose contents in the whole grain rice using LC-MS and GC-
MS
2. To study distribution patterns of micronutrients (Fe, Zn, Mn, P and Ca) in the rice
endosperm using inductively coupled plasma mass spectrometry (ICP-MS), and
desorption electrospray ionization (DESI) mass spectrometry
3. Genetic mapping to identify gene pool for metallomic and metabolomic directed
traits, and mainly about ion transporters

13. Methodology – Objective 1
“To investigate genotype based variation in polyphenols, vitamins, essential amino acids, fatty acids
lysopolysaccharides, and amylose contents in the whole grain rice using LC-MS and GC-MS”
1. Plant material:
 Seeds of high inorganic and organic nutrients density genotypes (Kalanamak, IR68144, etc.) will be
obtained form IRRI centers. Grains will be dehusked, and sets of brown and white rice will be prepared
for each genotype
2. LC-MS/MS based quantitative analysis of polyphenols, vitamins, essential amino acids,
and lysophospholipids:
 All the rice endosperm samples (brown and polished rice) will be milled into powder with a mixer, and
used for nutrient analysis
 Optimization extraction and LC-MS/MS quantitative methods for phenolic acids, flavonoids and
anthocyanins (Irakli et al, 2012; Shao et al., 2014)
Shao, et al. (2014). Journal of cereal science, 59(2), 211-218 ; . Irakli, et al. (2012).
Journal of separation science, 35(13), 1603-1611.

14. Methodology – Objective 1
“To investigate genotype based variation in polyphenols, vitamins, free amino acids, fatty acids and amylose contents
in the whole grain rice using LC-MS and GC-MS”
 Optimization of extraction and LC-MS/MS quantitative methods for water soluble vitamins (vitamin B1
(thiamin), vitamin B6 (pyridoxine), nicotinic acid, nicotinamide (B3) and vitamin C), and Vitamin E (CAO
et al. (2007); Yu et al. (2016))
 Optimization of extraction and LC-MS/MS quantitative method for free amino acids (Liyanaarachchi et
al. (2018))
 GC/GC-MS analysis of saturated and unsaturated fatty acids (Kasote et al., 2013)
 Optimization of extraction and LC-MS/MS quantitative method for lysophospholipids (Liu et al. (2014))
CAO et al. (2007). Food Science and Technology, 11; Yu, et al. (2016). Food chemistry, 197, 776-782;.
Kasote et (2013). Industrial crops and products, 42, 10-13.
Liu et al., (2014). Journal of agricultural and food chemistry, 62(28), 6600-6607.
Liyanaarachchi et al. (2018) Journal of Chromatography A, 1568, 131-139.

15. Methodology – Objective 2
“To study distribution patterns of micronutrients (Fe, Zn, Mn, P and Ca) in the rice endosperm using inductively coupled
plasma mass spectrometry (ICP-MS), and desorption electrospray ionization (DESI) mass spectrometry”
1. Elemental Analysis by inductively coupled plasma mass spectrometry (ICP-MS): Flour
samples will be digested initially, and then concentrations of elements (Mn, Fe, Zn, Mg, Ca and K) will
be determined using inductively coupled plasma mass spectrometry (Lu et al. (2013))
2. Distribution patterns of micronutrients using desorption electrospray ionization (DESI)
mass spectrometry: Sample preparation and desorption electrospray ionization (DESI) mass
spectrometric analysis will be carried out as per procedure described by Moore et al., 2014

Lu et al. (2013). PLoS One, 8(2), e57360;Moore et al. (2014). New Phytologist, 201(1), 104-115.

16. Methodology – Objective 3
“Genetic mapping to identify gene pool for metallomic and metabolomic directed traits, and mainly about ion
transporters
”
Genetic mapping for organic and inorganic nutrient traits including ion transporters traits
in rice grain: For genotyping, DNA will be extracted from leaf samples, and will be submitted to
Genotyping Services Laboratory of IRRI for genotyping Simultaneously, linkage mapping and QTL analysis
will be performed as per protocols described by Descalsota-Empleo et al. (2019)
Descalsota-Empleo et al. (2019). The Crop Journal.

17. Time Schedule of activities giving milestones through BAR diagramTime Schedule of activities giving milestones through BAR diagram

18. Expected Outcome
 Proposed study may provide detailed insight about organic and in-organic nutrients
composition rice endosperm of different genotypes, mainly, knowledge about co-
localization and co-deposition of organic and in-organic nutrients in rice endosperm
 Findings of rice endosperm imaging with DESI-MS will provide useful information
about differences in inorganic micronutrients loading in various species
 Results of proposed study may provide knowledge of gene pools involved in transport,
and loading of minerals within rice grains, which is important for understanding the
role of different elements in seed development, as well as for facilitating
biofortification of seed micronutrients in order to enhance seeds’ values in human
diets

19. "Rice Science For A Better World"