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Analysis of lysophospholipid content in low phytate rice mutants Chuan Tong, Yaling Chen, Yuanyuan Tan, Lei Liu, Daniel L.E. Waters, Terry Rose, Qing-yao Shu, and Jinsong Bao J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 13, 2017

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Journal of Agricultural and Food Chemistry

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Analysis of lysophospholipid content in low phytate rice mutants

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Chuan Tong1,2, Yaling Chen1, Yuanyuan Tan3, Lei Liu2,*, Daniel L. E. Waters2, Terry J.

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Rose2,4, Qingyao Shu3, Jinsong Bao1,*

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Zhejiang University, Huajiachi Campus, Hangzhou, 310029, China.

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2

Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology,

Southern Cross Plant Science, Southern Cross University, Lismore, NSW, 2480,

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Australia.

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3

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University, Hangzhou, 310029, China.

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4

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Australia.

Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang

Southern Cross Geoscience, Southern Cross University, Lismore, NSW, 2480,

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*Corresponding authors:

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Jinsong Bao, phone +86-571-86971932; fax +86-571-86971421; and emails:

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[email protected] (J. Bao).

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Lei

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[email protected] (L. Liu)

Liu,

phone

+61-02-6622-3211;

fax

+61-02-6622-3459;

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1

ACS Paragon Plus Environment

and

emails:


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Abstract

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As a fundamental component of nucleic acids, phospholipids and adenosine

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triphosphate, phosphorus (P) is critical to all life forms, however, the molecular

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mechanism of P translocation and distribution in rice grains are still not understood.

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Here, with the use of five different low phytic acid (lpa) rice mutants, the

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redistribution in the main P-containing compounds in rice grain, phytic acid (PA),

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lysophospholipid (LPL) and inorganic P (Pi), was investigated. The lpa mutants

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showed a significant decrease in PA and phytate-phosphorus (PA-P) concentration

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with a concomitant increase in Pi concentration. Moreover, defects in the OsST and

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OsMIK genes result in a great reduction of specific LPL components and LPL-

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phosphorus (LPL-P) contents in rice grain. In contrast, defective OsMRP5 and Os2-

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PGK genes led to a significant increase in individual LPLs components. The effect of

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the Os2-PGK gene on the LPLs accumulation was validated using breeding lines

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derived from a cross between KBNT-lpa (Os2-PGK mutation) and Jiahe218. This

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study demonstrates that these rice lpa mutants lead to the redistribution of Pi in

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endosperm and modify LPL biosynthesis. Increase in LPLs in endosperm in the lpa

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mutants may have practical applications in rice breeding to produce “healthier” rice.

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Keywords: Rice, phosphorus, phytic acid, lysophospholipids

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Abbreviations:

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AAC, apparent amylose content

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ANOVA, analysis of variance

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LPA, low phytic acid

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LPLs, lysophospholipids

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PA, phytic acids

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PA-P, phytate-phosphorus or phytic acid-phosphorus

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PHT and PT, phosphate transporter

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Pi, inorganic phosphorus

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Introduction

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As a fundamental component of nucleic acids, phospholipids (PLs) and

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adenosine triphosphate (ATP)1, phosphorus (P) is critical to all life forms. Three

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soluble phosphate forms, PO43-, HPO42- and H2PO4- can be absorbed by plants2,

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especially cereal crops. Remobilising most of plant vegetative P reserves to grains is a

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major driver of the global P cycle3.

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Phytic acid (PA), also known as phytate, or myo-inositol 1, 2, 3, 4, 5, 6-hexakis

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phosphate (InsP6), is the major storage form of phosphorus in seeds and cereal grain4.

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Up to 80% of total phosphorus in rice grain is stored as PA, but PA cannot be digested

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by monogastric animals including humans. In addition, PA is a strong chelator of

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divalent cations including Zn2+, Fe2+ and Ca2+, which inhibits their absorption in

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monogastric animals. Low phytate content grains are therefore desirable and this has

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led to efforts to decrease grain PA content through mutagenesis. Several low phytic

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acid (lpa) rice mutants have been reported5 which display either normal total

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phosphorus content with reduced PA-P and increased in Pi content, or increased

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levels of both Pi and myo-inositol phosphates containing five or fewer P esters5.

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Although PA has been the focus of attention because of its role as a storage form of P,

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PA and the intermediates of its synthesis have a myriad of functions including signal

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transduction, vesicle trafficking and polar auxin transport, biotic and abiotic stress

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response, nuclear function and regulation of phosphorus homeostasis, and hormonal

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signalling6.

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Several rice lpa genes have been isolated and their putative function identified.

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Rice lpa1-1 encodes a 2-phosphoglcerate kinase (PGK)7,8, and defects in lpa1-1

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results in a 45% reduction of grain PA content and a molar-equivalent increase in Pi

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content9. Other genes that have been isolated include a rice myo-inositol kinase

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(OsMIK)10, a myo-inositol 3-phosphate synthase (MIPS1) that results in an increase in

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available Pi and a corresponding molar-equivalent decrease in PA-P11. A single base

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pair change in a rice gene which encodes a multi-drug resistance-associated protein

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ABC transporter (OsMRP5), reduced 20% of PA content in grain12. In all rice lpa

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mutants, PA-P decreased and Pi increased, yet the impact of these mutations on the

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metabolism of other phosphorus containing entities was not reported.

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Phospholipids are important P-containing compounds in rice grain endosperm,

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especially their lyso forms (lysophospholipids, LPLs), which form amylose-lipid 4


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complexes

and

influence

rice

physicochemical

properties13,14.

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inclusion

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Lysophosphatidylcholine and lysophosphatidylethanolamine are the major rice starch

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LPLs and, depending on extraction methods, account for 30-40%15 or 48-67%16 of

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starch lipids. Rice endosperm LPL content ranges from 5610 to 9400 µg/g in non-

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waxy rice accessions and although the levels of specific LPLs were affected by

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environment and genotype × environment interactions, they were mainly determined

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by genetics17,18.

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Translocation of P from stems and leaves contributes substantially to grain P

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with 75% of total plant P being found in rice grains, primarily in the hull, aleurone

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and embryo19. Remobilization and redistribution of inorganic phosphorus (Pi) from

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rice hull, aleurone and embryo to endosperm would enhance the nutritional quality of

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rice for end-consumption, particularly if the redistribution was associated with

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decreased PA content. However, the pathways that mediate Pi remobilization and

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allocation within rice grain, such as from PA to PLs, are not known. We hypothesized

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the increased Pi that results from reduced PA synthesis in lpa rice mutants, could be

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transported into endosperm and enhance LPL biosynthesis. Individual LPLs were

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quantified in five lpa rice mutants and their respective wild genotypes to investigate if

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Pi could be reallocated to the endosperm and participate in biosynthesis of rice

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endosperm LPL. Furthermore, individual LPLs were quantified in nine inbreeding

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lines derived from the cross KBNT-lpa (Os2-PGK mutation) and Jiahe218 to validate

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the effect of lpa mutant on the rice endosperm LPLs accumulation. An improved

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comprehension of P metabolism in rice endosperm will inform manipulation and

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breeding improved rice cultivars.

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Materials and methods

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Plant materials

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Nine rice accessions including four wild type and five lpa mutants were used in

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this study (Table 1). The nine accessions were grown with two replications in the field

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in 2013 in Zhejiang University, Hangzhou, Zhejiang province, and in 2013-2014 in

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Lingshui, Hainan province, China. In addition, nine F7 breeding lines derived from a

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cross between KBNT-lpa and Jiahe218 (a japonica cultivar with low apparent

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amylose content) were grown along with their parents in the field in Lingshui, Hainan

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province in 2014-2015. In each replication, each rice accession was planted in three 5



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rows with six plants per row. The plant-to-plant spacing between and within rows was

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20 cm and 20 cm, respectively. Sowing was in late May and harvest in November in

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Hangzhou while sowing was in early December and harvest in April in Lingshui.

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Harvested rice grain were air-dried and stored at 4 oC until analysis. Rice grains were

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de-hulled to brown rice using a Satake Huller (Satake Co., Hiroshima, Japan). White

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rice grains were obtained from another Satake Rice Milling Machine (Satake Co.,

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Tokyo, Japan). Rice grains were ground to flour to pass through a 100 mesh sieve in a

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Cyclone sample mill (UDY Corporation, Fort Collins, Co., USA).

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Phytic acid content determination

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Phytic acid was analyzed using a modified method of Shi et al20. In brief, brown

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rice flour (25 mg) was placed into a 1.5 ml microcentrifuge and incubated in 1 ml 0.4

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M HCl for 3 hours at room temperature with continuous mixing. The extracts were

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centrifuged at 15,000 rpm for five minutes and 0.5 ml supernatant was transferred to a

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1.5 ml microcentrifuge. The extracted supernatant (15 µl) was added to 15 µl of

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deionized water and 30 µl 0.2 M HCl in triplicate in separate wells of a 96 well PCR

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plate. The PCR plate was centrifuged for 20 seconds at 300 rpm then 120 µl of

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ammonium iron (0.02%, w/v) sulphate-0.2 N HCl was added to each well. The PCR

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plate was re-centrifuged for 20 seconds at 300 rpm, capped, and then heated at 99 °C

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for 30 minutes. The PCR plate was cooled in an ice bath for 10 minutes and then

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centrifuged at 3,000 RCF for 30 minutes. For colorimetric determination, 80 µl of

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each sample and standard solution were transferred from the PCR plate into a 96 well

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plate (flat bottom) and 120 µl of 1% (w/v) 2,2’-bipyridine-1% (v/v) thioglycolic acid

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added and mixed for 10 minutes on a plate shaker. Finally, absorbance was measured

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at 519 nm with a KC4 multi-detection microplate reader (Bio-Tek Instruments, USA)

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in triplicate.

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Inorganic phosphorus (Pi) content determination

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Pi was analyzed using a modified protocol of Kitson and Mellon21. In brief,

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approximately 25 mg brown or milled rice flour was digested with 1 ml 0.4 M HCl in

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a 1.5 ml microfuge tube for 3 hours at room temperature with continuous mixing.

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Each sample was centrifuged at 15,000 rpm for 5 minutes then 70 µl of the

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supernatant was mixed with 40 µl of deionized water in a 96 well plate (flat bottom),

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in triplicate. For colorimetric determination, 50 µl vanadate (0.11%, m/v) and 40 µl 6


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molybdate (1.25%, m/v) were added into each well and mixed for 10 minutes on a

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plate shaker and absorbance measured at 460 nm.

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Starch phospholipid extraction and HPLC-MS determination

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A single step extraction method was used to extract rice endosperm

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phospholipids18. Briefly, approximately 16 mg of milled rice flour was extracted with

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0.8 ml of 75% n-propanol (v/v) at 100 oC for 2 h, in triplicate. Vials were weighed

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before and after extraction to calculate solvent loss. All vials were cooled, centrifuged

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at 3,000 rpm for 7 minutes and 0.5 ml supernatant removed into a 2.0 ml vial for LC-

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MS analysis. Lysophospholipid content was determined by HPLC-MS according to

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Liu et al18.

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Genotyping of breeding lines The CAPS maker (Table S1)22 and microsatellite23 was used to diagnose the genotype of nine breeding lines derived from KBNT-lpa and Jiahe218.

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Statistical Analysis

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Data analyses were performed with the SAS System Edition for Windows

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version 9.1 (SAS Institute Inc., Cary, NC, USA). Analysis of variance (ANOVA) was

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performed with Procglm. Means of genotypes and environments was conducted with

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PROC means, followed by Duncan’s multiple comparisons (P< 0.05).

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Results

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Phytic acid and P content of lpa rice mutants

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All mutant lines had significantly lower PA and PA-P content than their

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respective wild types. PA content ranged from 14.2 to 18.1 mg/g for wild types and

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8.9 mg/g to 13.4 mg/g in mutant genotypes over 2 years (Table 2). PA-P decreased

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from 4.0-5.1 mg/g to 2.5-3.8 mg/g in brown rice (Table 2). The reductions represented

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PA decreases of 28.0-36.9%, 37.8-40.3%, 9.6-23.9% and 25.7-39.4% in OsST,

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OsMIK, OsMRP5 and Os2-PGK mutants, respectively (Table 2). Wild type Pi content

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varied from 1.1 to 1.7 mg/g in brown rice and from 0.52 to 0.87 mg/g in milled rice

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(Table 2) while lpa rice cultivars had higher Pi content than brown rice (1.7-3.5 mg/g)

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and milled rice (1.0-1.3 mg/g) (Table 2).

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LPL-phosphorus (LPL-P) content in rice endosperm declined from 0.33-0.61

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mg/g to 0.28-0.47 mg/g due to OsST mutations and 0.49 mg/g to 0.42-0.47 mg/g from

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OsMIK gene defects while the LPL-P content in cultivars J2 and KBNT-lpa increased

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from 0.49 mg/g to 0.53-0.54 mg/g and from 0.63 to 0.71-0.72 mg/g, respectively

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(Table 2). LPL-P decreased 10.6-31.4% in OsST and 3.9-14.7% in OsMIK mutants

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compared to their respective wild types, whereas LPL-P increased by 8.2-10.7% in

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OsMRP5 and 12.8-14.8% in Os2-PGK mutants compared to their respective wild

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types (Table 2).

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Endosperm LPLs in lpa rice mutants

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LPCs and LPEs detected were LPC14:0, LPC16:0, LPC18:1, LPC18:2,

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LPC18:3, and LPE14:0, LPE16:0, LPE18:1, LPE18:2, LPE18:3, total LPC (TLPC)

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(where TLPC = LPC14:0 + LPC16:0 + LPC18:1 + LPC18:2 +LPC18:3), total LPE

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(TLPE) (where TLPE = LPE14:0 + LPE16:0 + LPE18:1 + LPE18:2 +LPE18:3) and

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total LPLs (TLPL) (where TLPE = TLPC + TLPE).

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Rice endosperm LPC component variation between genotype, season and

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genotype × season were detected (P < 0.001) and accounted for 72.9-98.2%, 0.1-1.9%

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and 1.4-23.7% of the total variance, respectively (Table 3). The effects of genotype ×

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season interaction for LPE components were also significant (P < 0.001) (Table 3)

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with variation due to genotype accounting for a large proportion of the total variance

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(87.2-98.3%), whereas season and genotype × season interaction accounted for only

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0.4-1.6% and 0.7-8.7% of total variance, respectively (P < 0.001) (Table 3). Total

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LPL content was strongly influenced by genotype (P < 0.001), accounting for 97.5%

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of the total variance (Table 3).

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Extensive variation in LPL composition was observed among the nine rice

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accessions (Table 4). TLPC and TLPE ranged from 4047 µg/g to 8775 µg/g and 585

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µg/g to 2567 µg/g, accounting for 77-87% and 13-23% of total LPLs, respectively.

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LPC16:0, LPC18:2 and LPE16:0 were the main individual LPL components, which

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varied from 2846 to 5507 µg/g, 714 to 1822 µg/g and 400 to 1778 µg/g, and

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accounted for 46-61%, 15-21% and 9-16% of total LPLs, respectively (Table 4).

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Minor components involved LPC14:0, LPC18:1, LPC18:3, LPE14:0, LPE18:1,

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LPE18:2 and LPE18:3, which represented 2.9-5.5%, 6.3-12.5%, 0.2-0.5%, 0.3-0.9%, 8


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0.7-1.7%, 2.9-5.0% and < 0.1% of total LPLs, respectively (Table 4).

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LPA mutant accessions dramatically differed from the wild type in individual

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LPL components. All individual LPLs decreased in OsST mutants with reductions of

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9.0-15%, 20-31% and 37-38% for the main constituents of LPC16:0, LPC18:2 and

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LPE16:0, respectively, and 5.0-50% for other minor LPL components. A 16-23%

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decrease of TLPL resulted from 13-20% in TLPC and 35-37% decrease in TLPE (P

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