Received 3 Feb. 2007 Accepted 3 Jul. 2007
Relationships between Phosphatidylglycerol Molecular Species of Thylakoid Membrane Lipids and Sensitivities to Chilling-induced Photoinhibition in Rice (Oryza sativa L.)
Su-Qin Zhu 1, 2, 3, Hua Zhao 1, 2, Jian-Sheng Liang3, Ben-Hua Ji*[1], 2 and De-Mao Jiao 4
（1. Institute of Bioengineering, Nantong University, Nantong 226007, China;
2. College of Life Sciences, Nantong University, Nantong 226007, China;
3. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225000, China;
4. Institute of Agrobiological Genetics and Physiology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China）
Abstract
In an attempt to explore the relationships between phosphatidylglycerol (PG) molecular species of thylakoid membrane lipids and sensitivities to chilling-induced photoinhibition, PG molecular species, D1 protein, electron transport activities of thylakoid membrane and the potential quantum yield (Fv/Fm) in rice treated under middle and low PFD at 11 ℃ were analysed by high performance liquid chromatography (HPLC), enzyme hydrolysis, gas phase chromatography (GC) and so on. Results showed that the major molecular species of PGs in rice thylakoid membrane were 18:3/16:0, 18:3/16:1(3t), 18:2/16:0, 18:2/16:1(3t), 18:1/16:0, 18:1/16:1(3t), 16:0/16:0, 16:0/16:1(3t). There were large differences in contents of unsaturated PG molecular species such as 18:1~3/16:0~16:1(3t) and saturated PG molecular species like 16:0/16:0~16:1(3t) among japonica cv 9516 (j-9516), japonica-indica hybrid F1 j-9516/i-SY63 (ji-95SY) and indica cv Shanyou 63 (i-SY63). J-9516 containing higher contents of unsaturated PG molecular species was manifest in stable D1 protein contents under chill and tolerant to chill-induced photoinhibition. In contrast to j-9516, i-SY63 with lower contents of unsaturated PG molecular species, exhibited unstable D1 protein contents under chill and was sensitive to chill-induced photoinhibition. ji-95SY containing middle contents of unsaturated PG molecular species between those of j-9516 and i-SY63, exhibited mid extent of sensitivity to chill-induced photoinhibition. The loss in D1 protein has been also accounted for the inhibition in electron transport activity of thylakoid membrane and the observed decline in Fv/Fm. The PG molecular species efficient in raising chilling-resistant capacity were those containing unsaturated fatty acids, namely, unsaturated PG molecular species. These results implied that the substrate selectivity of the glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts towards 16:0 or 18:1 and the activity of fatty acid desaturases displayed greatly difference between japonica and indica rice. It was possible to enhance the capacity of resistance to chilling-induced photoinhibition by improving or modifying GPAT gene.
Keywords: chilling-induced photoinhibition; D1 protein; molecular species; phosphatidylglycerol; rice.
Zhu SQ, Zhao H, Liang JS, Ji BH, Jiao DM (2007). Relationships between phosphatidylglycerol molecular species of thylakoid membrane lipids and sensitivities to chilling-induced photoinhibition in rice (Oryza sativa L.). J Integr Plant Biol
Available online at www. Blackwell-synergy. Com/links/toc/jipb, www. Jipb. net
Thylakoid membranes in chloroplasts are the sites of light absorption, electron transfer, photosynthetic energy conversion, the decomposition of water under light and the release of oxygen (Aldridge et al. 2005). Lipid molecules constitute the backbone of membrane, however, the thylakoid membrane consists of mostly galactolipid is different absolutely from biological membranes of non-photosynthetic apparatus such as the cell membrane of animal or bacteria which are composed almost of phospholipid. In thylakoid membrane, glycolipids are composed of monogalactosyldiglyceride (MGDG) mainly and secondly digalactosyldiglyceride (DGDG). They account for 70% (mol %) of total membrane lipids. In addition, thylakoid membrane still contains own thiolipid-----an anionic lipid of sulfoquinovosyldiglyceride (SQDG), which is rare in non-photosynthetic tissue. Phospholipid in thylakoid membrane is only an anionic lipid of phosphatidylglycerol (PG) in higher plant chloroplasts with relative contents of 5%-15%. PGs occur widely almost all functional parts in thylakoid membrane (Webb et al. 1991). Researchers have studied many biological functions of this phospholipid. Genetic, biochemical, and structural studies of photosynthetic organisms have revealed that PGs are crucial to the photosynthetic transport of electrons, the development of chloroplasts, and tolerance to chilling. Evidences (Jordan et al. 2001) have disclosed a specific binding of PG molecules with photosynstemⅡ (PSⅡ), PGs play an important role in binding extrinsic proteins required for sustaining a functional Mn cluster on the donor side of PSII (Sakurai et al. 2007). Studies show that either charge separation or electron transfer in thylakoid membrane depends primarily on the presence of PGs (Sakurai et al. 2006; Sakurai et al. 2003). A special PG molecular species with the trans-3-hexadecenoicacid (16:1(3t)) at the sn-2 position of its glycerol backbone is considered to play an important role in the ordered assembly and structural maintenance of the photosynthetic apparatus such as polymer of PSⅡ（Kruse et al. 2000）, PSⅠ light-harvesting pigment protein complex (LHCⅠ) (Schmid et al. 1997), PSⅡ light-harvesting pigment protein complex (LHCⅡ) (Sakurai et al. 2006), and the optimal conformation of D1 protein in PSⅡ reaction center (Sakurai et al. 2003). Studies on membrane functions of mutants defective in some particular lipid further exhibit that PGs are the irreplaceable composition for photosynthetic apparatus (Sato et al. 2000).
PGs in thylakoid membrane, containing more saturated fatty acids and a special trans-3-hexadecenoic acid (16:1(3t)) and exhibiting firstly phase transition in process of dropping temperature, are the most sensitive to chilling temperature and considered as the important molecule to affect membrane fluidity (Murata et al. 1983). PGs are the important phospholipid related to photosynthetic activity and chilling-resistance in plants. Obvious correlation between saturated levels of fatty acids in PG molecular and photosynthetic chilling-resistance were obtained and validated in transgenic plants by Murata et al (1992).
Japonica rice, with the thylakoid membrane lipids containing more unsaturated fatty acids, is tolerant to chilling-induced photoinhibition. Whereas indica rice, with the thylakoid membrane lipids containing more saturated fatty acids, is sensitive to chilling-induced photoinhibition (Ji Benhua, 2003). Report (Yang et al, 1994) showed that the phase transition temperature of PG in dry embryo membrane lipids was related to chilling-resistance in rice.
The major target of our research efforts is to establish molecular foundation for transgenic technology of chilling- and photoinhibition-resistance in rice. In this study, our attention is focused on the differences of PG molecular species composition in thylakoid membrane among japonica rice, indica rice and their F1 hybrids. Another line of our research is focused on the relationships between PG molecular species of thylakoid membrane lipids and sensitivities to chilling-induced photoinhibition in rice by high performance liquid chromatography (HPLC), enzyme-catalysed degradation and thin layer chromatography (TLC).
Results
Changes in PSⅡ-D1 protein contents under medium and lower PFD and chilling
Under natural conditions (0d), PSⅡ-D1 protein contents were about 1.91 μmol mg-1· Chl and no obvious difference among j-9516, ji-95SY and i-SY63 were obtained. However, under a PFD of 600μmol m-2· s-1 at 11 °C D1 protein contents decreased continuously with the delaying of treatment time, and about 71.3%, 66.0% and 56.3% in j-9516, ji-95SY and i-SY63, respectively, on the 5th day. D1 protein contents declined more in i-SY63 than in j-9516, and the extent of D1 protein contents in ji-95SY was between those in j-9516 and i-SY63, but bias to maternal line japonica (Fig. 1). Furthermore, under 100 μmol m-2· s-1 at 11 °C, D1 protein contents decreased to 83.3%, 76.4% and 69.1% on the 3rd day, then climbed slowly to 92.2%, 83.0% and 72.8% in j-9516, ji-95SY and i-SY63, respectively on the 5th day. Compared with i-SY63, D1 protein contents in j-9516 decreased in a narrow range during the first 3 days then climbed in a greater extent after 3 days. The changes of D1 protein contents in ji-95SY were also between the changes in j-9516 and i–SY63, and still bias to maternal line japonica (Fig 1).
Fig. 1 Changes of PSⅡ-D1 protein contents of thylakoid membrane in j-9516, ji-95SY and i-SY63 under middling (600 μmol m-2· s-1) and lower (100μmol m-2· s-1) light at 11 °C.Changes of PG molecular species composition in rices under medium and lower PFD and chilling
There were generally sixteen-carbon saturated fatty acids (16:0) or some eighteen-carbon unsaturated fatty acids (18:1, 18:2, 18:3) at sn-1 position, and always trans-3-hexadecenoic acid (16:1(3t)) and sixteen-carbon saturated fatty acid (16:0) at sn-2 position of glycerol backbone in PG molecular. This was consistent with the investigation of Frentzen M et al (1987). Different distribution and combination of fatty acids at sn-1 or sn-2 position of glycerol backbone formed diversified PG molecular species. The major molecular species (represented by combinations of abbreviated forms of fatty acids at sn-1 and sn-2 position, e.g. 18:3/16:0, sn-1-18:3-sn-2-16:0) were 18:3/16:0 PG, 18:3/16:1(3t) PG, 18:2/16:0 PG, 18:2/16:1(3t) PG, 18:1/16:0 PG, 18:1/16:1(3t) PG, 16:0/16:0 PG, 16:0/16:1(3t) PG. The level of each PG molecular species changed during chilling-treatment time. These changes depended on rice lines. With the exception of 18:2/16:0~16:1(3t) PGs, the relative contents of 18:3/16:0~16:1(3t), 18:1/16:0~16:1(3t) or 16:016:0~16:1(3t) PGs were great difference between j-9516 and i-SY63, and were correlated with chilling-induced photoinhibition of rice. There were, for example, about 34.3 mol %s and 27.6 mol %s of 18:3/16:0~16:1(3t) PGs, about 25.9 mol %s and 15.8 mol %s of 18:1/16:0~16:1(3t) PGs, and about 12.2 mol %s and 28.6 mol %s of 16:0/16:0~16:1(3t) PGs in j-9516 and i-SY63, respectively. The mol %s of PGs in ji-95SY were between the values of two parents.
Fig.2. Changes in the composition of PG molecular species (represented by combinations of abbreviated forms of fatty acids at sn-1 and sn-2 position) in thylakoid membrane of j-9516 (upper), ji-95SY (mid) and i-SY63 (lower) under medium (600 μmol m-2 · s-1, left) and lower (100μmol m-2·s-1, right) light.Results showed that temperatures altered mainly the levels of 18:3/16:0~16:1(3t) PG, 18:1/16:0~16:1(3t) PG and 16:0/16:0~16:1(3t) PG. Under 600 μmol m-2∙ s-1 and chilling (11 °C) conditions, mol %s of unsaturated PG molecular species such as 18:3/16:0~16:1(3t) PGs, 18:1/16:0~16:1(3t) PGs decreased continuously, from 34.3 mol%, 31.1 mol% and 27.6 mol% on the 0 day to 30.1 mol%, 23.8 mol% and 17.3 mol% on the 5th day in j-9516, ji-95SY and i-SY63, respectively (Fig. 2A, B, C). In contrast, the mol %s of 16:0/16:0 PG~16:1(3t) PG species increased from 12.2 mol%, 20.3 mol% and 28.6 mol% on the 0 day to 19.6 mol%, 30.8 mol% and 42.3 mol% on the 5th day in j-9516, ji-95SY and i-SY63, respectively (Fig. 2A, B, C). Furthermore, under a PFD of 100 μmol m-2∙ s-1 and chilling (11 °C), the mol %s of 18:3/16:0~16:1(3t) PGs molecular species decreased from 34.3 mol%, 30.9 mol% and 27.6 mol% on the 0 day to 30.8 mol%, 25.1 mol% and 19.1 mol% on the 3rd day, then started to increase to 32.6 mol%, 28.8 mol% and 20.4 mol% on the 5th day in j-9516, ji-95SY and i-SY63, respectively (Fig. 2D, E and F). whereas, the mol %s of 16:0/16:0~16:1(3t) PG molecular species increased from 12.2 mol%, 20.3 mol% and 28.6 mol% on the 0 day to 17.7 mol%, 28.5 mol% and 39.5 mol% on the 3rd day, then started to decrease to 15.6 mol%, 25.1 mol% and 37.4 mol% on the 5th day in j-9516, ji-95SY and i-SY63, respectively (Fig. 2D, E and F). These results clearly showed that both japonica and indica are able to adjust their PG molacular species composition to adapt the changes of lower temperature.
For the sake of explaining clearly, the unsaturated level of fatty acid (ULFA) of PG molecular species was defined as follows: ULFA = (18:1) mol % + (18:2) mol % × 2 + (18:3) mol % × 3. Investigations showed that ULFAs were different among three lines. Under natural conditions (0 day) ULFAs were about 186.3%, 168.8% and 153.8% in j-9516, ji-95SY and i–SY63, respectively. Nevertheless, under a PFD of 600 μmol m-2· s-1 and chilling (11 °C), the values of ULFA reduced continuously with the delaying of treatment time and to 165.5%, 141.6% and 117.3% on the 5th day in j-9516, ji-95SY and i–SY63, respectively (Fig.3 left). When, under a PFD of 100 μmol m-2∙s-1 and chilling (11 °C), ULFAs reduced to their lowest values about 169.1 %, 147.1 % and 122.4 % on the 3rd day, then started to increased to 175.0 %, 157.5 % and 129.3 % on the 5th day in j-9516, ji-95SY and i–SY63, respectively (Fig.3 right).
Fig.3 Changes in unsaturated level of fatty acids (ULFA) of PG molecules of thylakoid membrane in j-9516, ji-95SY and i-SY63 under medium (600 μmol m-2 s-1) and lower (100 μmol m-2 s-1) light at 11 °C.ULFA% = (18:1) mol % + (18:2) mol % × 2 + (18:3) mol % × 3
The changes of electron transport activities and the potential quantum yield of PSII (Fv/Fm) in rices under medium and lower PFD and chilling.
Since the chilling stress caused the decrease of D1 protein contents, we further analyzing the PSⅡ activities. Evidence showed that the loss in D1 protein had been accounted for the inhibition in PSII activity and also the observed decline in Fv/Fm was caused by the degradation of D1 protein. PSⅡ electron transport activities (H2O→DCIP) and the potential quantum yield of PSII (Fv/Fm) all decreased continuously during the treatment time under a PFD of 600 μmol m-2∙s-1 and chilling (11 °C). However under a PFD of 100 μ mol m-2 ∙ s-1 and chilling (11℃) PSⅡ electron transport activities (H2O→DCIP) and Fv/Fm all decreased continuously during first 3 days, then increased slowly (Fig. 4). The trends of change were identical with that of D1 protein content. Making a comparison among three rice lines, we found that PSⅡ electron transport activities (H2O→DCIP) and Fv/Fm changed in a narrow range in j-9516, and a great range in i-SY63. It was indicated that the combination of light and low temperature appeared to change the composition of PG molecular species and further inactivate PSII activities very rapidly as a consequence of their synergistic effects. PG molecular species played an important role in response of thylakoid membrane to chilling-induced photoinhibition in rice.
Fig. 4 Changes of electron transport activities (ETA) and the potential quantum yield of PSII (Fv/Fm) in j-9516, ji-95SY and i-SY63 under medium (600 μmol m-2 s-1) and lower (100 μmol m-2 s-1) light at 11 °C.Discussion
Effects of compositions of PG molecular species on sensitivity to chilling-induced photoinhibition in rice
Lipid unsaturation, the major factor to maintain thylakoid membrane fluidity, is affected by temperature. In this work, we analysed the molecular species composition of PG in thylakoid membranes under the chilling conditions to investigate how the compositions of PG molecular species affects the sensitivity of rice to chilling. Of the 8 molecular species detected, temperature affected mainly the relative contents of unsaturated PG molecular species and saturated PG molecular species. Under a PFD of 600 μmol m-2· s-1 at 11 °C (Fig. 2), for example, the contents of saturated PG molecular species (16:0/16:0~16:1(3t)) from about 12.2 mol% and 28.6 mol% of 0 d (control), with a difference value of 16.4 mol%, become to 19.6 mol% and 42.3 mol% of the 5th day, with a difference value of 22.7 mol% in thylakoid membrane lipids of japonica and indica rice, respectively. This is called photoinhibition of photosynthesis. Whereas under a PFD of 100 μmol m-2· s-1 at 11 °C (Fig 2), the contents of saturated PG molecular species from the same values of 0 d increased to 17.7 and 39.5 mol% of the 3rd day with a difference value of 21.8 mol%, then decreased slowly to 16.5 and 37.4 mol% of the 5th day with a difference value of 20.9 mol% in thylakoid membrane lipids of japonica and indica rice, respectively. The mol %s of saturated PG molecular species in japonica-indica hybrid F1 were the middle values between the values of indica and japonica rice. This is called acclimation of photosynthesis (Morgan-Kiss et al. 2006). In contrast to the contents of saturated PG molecule species, either under a PFD of 600 μmol m-2· s-1 at 11 °C or under a PFD of 100 μmol m-2· s-1 at 11 °C, the contents of unsaturated PG molecule species (18:1~18:3/16:0~16(3t)) changed reversely. Analysing the compositions of PG molecular species from different rice lines showed that decrease in unsaturated level of fatty acids of PGs was accompanied by a reduction in the contents of D1 protein, and consistent with this was the fact that, the rice with higher contents of unsaturated PG molecules such as j-9516 exhibited higher chilling-resistant capacity than the rice with higher contents of saturated PG molecules such as i-SY63. There was a positive correlation between unsaturated level of fatty acids in PGs and D1 protein contents, or a negative correlation between the mol %s of saturated PG moleculars and D1 protein contents (Fig. 5). So, chilling sensitivity of rice was closely correlated with the contents of unsaturated PG molecular species (Fig. 5).