Lipidome Profiling of Phosphorus Deficiency-Tolerant Rice Cultivars Reveals Remodeling of Membrane Lipids as a Mechanism of Low P Tolerance.

Plants have evolved various mechanisms for low P tolerance, one of which is changing their membrane lipid composition by remodeling phospholipids with non-phospholipids. The objective of this study was to investigate the remodeling of membrane lipids among rice cultivars under P deficiency. Rice (Oryza sativa L.) cultivars (Akamai, Kiyonishiki, Akitakomachi, Norin No. 1, Hiyadateine, Koshihikari, and Netaro) were grown in 0 (-P) and 8 (+P) mg P L-1 solution cultures. Shoots and roots were collected 5 and 10 days after transplanting (DAT) in solution culture and subjected to lipidome profiling using liquid chromatography-mass spectrometry. Phosphatidylcholine (PC)34, PC36, phosphatidylethanolamine (PE)34, PE36, phosphatidylglycerol (PG)34, phosphatidylinositol (PI)34 were the major phospholipids and digalactosyldiacylglycerol (DGDG)34, DGDG36, 1,2-diacyl-3-O-alpha-glucuronosylglycerol (GlcADG)34, GlcADG36, monogalactosyldiacylglycerol (MGDG)34, MGDG36, sulfoquinovosyldiacylglycerol (SQDG)34 and SQDG36 were the major non-phospholipids. Phospholipids were lower in the plants that were grown under -P conditions than that in the plants that were grown under +P for all cultivars at 5 and 10 DAT. The levels of non-phospholipids were higher in -P plants than that in +P plants of all cultivars at 5 and 10 DAT. Decomposition of phospholipids in roots at 5 DAT correlated with low P tolerance. These results suggest that rice cultivars remodel membrane lipids under P deficiency, and the ability of remodeling partly contributes to low P tolerance.

Ancient rice cultivar extensively replaces phospholipids with non-phosphorus glycolipid under phosphorus deficiency.

Recycling of phosphorus (P) from P-containing metabolites is an adaptive strategy of plants to overcome soil P deficiency. This study was aimed at demonstrating differences in lipid remodelling between low-P-tolerant and -sensitive rice cultivars using lipidome profiling. The rice cultivars Akamai (low-P-tolerant) and Koshihikari (low-P-sensitive) were grown in a culture solution with [2 mg l-1 (+P)] or without (-P) phosphate for 21 and 28 days after transplantation. Upper and lower leaves were collected. Lipids were extracted from the leaves and their composition was analysed by liquid chromatography/mass spectrometry (LC-MS). Phospholipids, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidylinositol (PI), lysophosphatidylcholine (lysoPC), diacylglycerol (DAG), triacylglycerol (TAG) and glycolipids, namely sulfoquinovosyl diacylglycerol (SQDG), digalactosyldiacylglycerol (DGDG), monogalactosyldiacylglycerol (MGDG) and 1,2-diacyl-3-O-alpha-glucuronosyl glycerol (GlcADG), were detected. GlcADG level was higher in both cultivars grown in -P than in +P and the increase was larger in Akamai than in Koshihikari. DGDG, MGDG and SQDG levels were higher in Akamai grown in -P than in +P and the increase was larger in the upper leaves than in the lower leaves. PC, PE, PG and PI levels were lower in both cultivars grown in -P than in +P and the decrease was larger in the lower leaves than in the upper leaves and in Akamai than in Koshihikari. Akamai catabolised more phospholipids in older leaves and synthesised glycolipids in younger leaves. These results suggested that extensive phospholipid replacement with non-phosphorus glycolipids is a mechanism underlying low-P-tolerance in rice cultivars.

Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogon.

Cultivated rice (Oryza sativa) possesses various labdane-related diterpene synthase genes, homologs of ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins.

Objective evaluation of whiteness of cooked rice and rice cakes using a portable spectrophotometer.

The whiteness of cooked rice and rice cakes was evaluated using a portable spectrophotometer with a whiteness index (WI). Also, by using boiled rice for measurement of Mido values by Mido Meter, it was possible to infer the whiteness of cooked rice without rice cooking. In the analysis of varietal differences of cooked rice, 'Tsuyahime', 'Koshihikari' and 'Koshinokaori' showed high whiteness, while 'Satonoyuki' had inferior whiteness. The whiteness of rice cakes made from 'Koyukimochi' and 'Dewanomochi' was higher than the whiteness of those made from 'Himenomochi' and 'Koganemochi'. While there was a significant correlation (r = 0.84) between WI values and whiteness scores of cooked rice by the sensory test, no correlation was detected between the whiteness scores and Mido values, indicating that the values obtained by a spectrophotometer differ from those obtained by a Mido Meter. Thus, a spectrophotometer may be a novel device for measurement of rice eating quality.

Reverse-genetic approach to verify physiological roles of rice phytoalexins: characterization of a knockdown mutant of OsCPS4 phytoalexin biosynthetic gene in rice.

A variety of labdane-related diterpenoids, including phytocassanes, oryzalexins and momilactones, were identified as phytoalexins in rice (Oryza sativa L.). Momilactone B was also isolated as an allelochemical exuded from rice roots. The biosynthetic genes of these phytoalexins have been identified, including six labdane-related diterpene cyclase genes such as OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8 and OsKSL10. Here we identified an OsCPS4 knockdown mutant, cps4-tos, by screening Tos17 mutant lines using polymerase chain reaction. OsCPS4 encodes a syn-copalyl diphosphate synthase responsible for momilactones and oryzalexin S biosynthesis. Because Tos17 was inserted into the third intron of OsCPS4, the mature OsCPS4 mRNA was detected in the cps4-tos mutant as well as the wild type. Nevertheless, mature OsCPS4 transcript levels in the cps4-tos mutant were about one sixth those in the wild type. The cps4-tos mutant was more susceptible to rice blast fungus than the wild type, possibly due to lower levels of momilactones and oryzalexin S in the mutant. Moreover, co-cultivation experiments suggested that the allelopathic effect of cps4-tos against some kinds of lowland weeds was significantly lower than that of the wild type, probably because of lower momilactone content exuded from cps4-tos roots. A reverse-genetic strategy using the cps4-tos mutant showed the possible roles of momilactones not only as phytoalexins but also as allelopathic substances.

Relative abundance of Delta(5)-sterols in plasma membrane lipids of root-tip cells correlates with aluminum tolerance of rice.

We investigated variations in aluminum (Al) tolerance among rice plants, using ancestor cultivars from the family line of the Al-tolerant and widely cultivated Japonica cultivar, Sasanishiki. The cultivar Rikuu-20 was Al sensitive, whereas a closely related cultivar that is a descendant of Rikuu-20, Rikuu-132, was Al tolerant. These two cultivars were compared to determine mechanisms underlying variations in Al tolerance. The sensitive cultivar Rikuu-20 showed increased permeability of the plasma membrane (PM) and greater Al uptake within 1 h of Al treatment. This could not be explained by organic acid release. Lipid composition of the PM differed between these cultivars, and may account for the difference in Al tolerance. The tolerant cultivar Rikuu-132 had a lower ratio of phospholipids to Delta(5)-sterols than the sensitive cultivar Rikuu-20, suggesting that the PM of Rikuu-132 is less negatively charged and less permeabilized than that of Rikuu-20. We used inhibitors of Delta(5)-sterol synthesis to alter the ratio of phospholipids to Delta(5)-sterols in both cultivars. These inhibitors reduced Al tolerance in Rikuu-132 and its Al-tolerant ancestor cultivars Kamenoo and Kyoku. In addition, Rikuu-132 showed a similar level of Al sensitivity when the ratio of phospholipids to Delta(5)-sterols was increased to match that of Rikuu-20 after treatment with uniconazole-P, an inhibitor of obtusifoliol-14alpha-demethylase. These results indicate that PM lipid composition is a factor underlying variations in Al tolerance among rice cultivars.