29. Leaf proteins of PGMR during photoperiod-sensitive stage of fertility transformation
X.Z. BI1, Y.H. XIAO1 and Y.B. TAO 2
1) College of Life Sciences, Wuhan University. Wuhan. 430072
2) Department of Biology, Huanggang Teacher's College. Huanggang, Hubei, 436100 P.R. China
Photoperiod-sensitive genic male-sterile rice (PGMR) Nongken 58s (i.e. 58s) is a mutant of Oryza sativa L. subs. japonica rice Nongken 58 (i.e. 58) which was first found by Shi (1985). It has the characteristic of fertility alteration regulated by photoperiod, which is controlled by one or two recessive nuclear gene(s) (Wang et al. 1991). Proteins are not only products of gene expression but also the executors of many important physiological functions. So, studies on leaf proteins of Nongken 58s during photoperiod-sensitive stage of fertility transformation arc helpful for revealing the molecular mechanisms of photoperiod-sensitive male sterility.
When young panicles of 58s and 58 were at secondary branch primordium differentiation (III) stage, plants were divided into two groups: one was kept under short-day (SD) with daily average temperature 26.8°C (light 27.1°C, dark 26.4°C), the other was exposed to 14h long-day (LD, 10h natural light supplemented with 4h artificial light of 1500Lx) with daily average temperature 26.8°C (light 27.4°C, dark 26.1°C). The plants were grown under natural long daylight during the period from the pollen meiotic division stage (VI) to heading time. In differentiation stage of secondary branch primordium (III), early stage of pistil and stamen formation (IV) (the 1st day), mid stage of pistil and stamen formation (the 3rd day) and formation stage of pollen mother cell (V), the unfolded second complete leaves of main stems under different photoperiod conditions were collected. Tillers were left for examining fertility later.
Furthermore, during the stages from III to V, all unfolded functional leaves (3-4 in total) of main stem were daubed with distilled water and 20ppm cycloheximide (CHX). In addition, 20ppm CHX was sprayed onto leaves of the main stalk in LD and SD at stage IV. The pollen fertility was examined, and data on self-fertility were collected after the plants matured. Protein extraction and two-dimensional gel electrophoresis were carried out following the system modified by Yang et al. (1993). Coomassie brilliant blue R-250 was used for protein staining.
About 270 components could be detected in mature functional leaves during photoperiod-sensitive stage of fertility transformation by the modified electrophoresis system. Their MW and pI were 10-95kD and pH 3.5-8.8 respectively. Almost all the proteins remained relatively stable between different materials and different photoperiodic treatments. A few proteins, however, varied at different development stages of young panicle under the influence of photoperiods. At least five leaf protein components in PGMR 58s varied with the photoperiod at the special development stage during photoperiod-sensitive stage of fertility transformation, and were thus related to fertility transformation. Some components existed only in leaves of 58s LD but did not occur in leaves of 58s SD, 58 SD and 58LD. These special proteins regulated by photoperiod, such us spot [MW53.7kD, pI6.5] of leaves at stage IV (the 3rd day) and spot [MW59.6kD. pI5.9] of leaves at stage V. may be, related to the expression of male-sterility. Therefore, these components might be the specially expressed products of photoperiod-sensitive genie male-sterile gene. Some other protein components such as spots [MW60.3kD, pI7.3], [MW60.3kD, pI7.0] and [MW19.5kD, pI3.51 at the stage IV (the 3rd day) were not present under sterile condition (58s LD) but appeared under fertile condition. This suggests the lack of some functional proteins during male-sterility expression. Thus, it could be inferred that, in the special genetic background, LD inhibited the expression of some genes related to normal fertility, while SD induced their expression, resulting in the final fertility differences.
In order to explore the biosynthesis of special proteins related to fertility transformation regulated by photoperiod, and verify the relationship between the special protein and fertility transformation, the leaves of 58s were treated with CHX, at photoperiod-sensitive stages. As CHX is a special inhibitor of cytoplasmic protein synthesis, this treatment could test the reliability of the above inference. In LD sterile condition, the percent of both pollen and self fertility in the control were 0. While the leaf surface was badly damaged after treatment with CHX, but the pollen fertility restored to 11.33%. But the pollen fertility restoration was not high enough, and the percent of self fertility was still 0. It's interesting that the pollen fertility of 58s in LD treated 3 times with CHX also reached to 9.25% at the most sensitive stage (IV). It indicated primarily that inhibitor of protein synthesis CHX was somewhat effective for restoring pollen fertility of 58s under sterile condition. This result was consistent with that of treatment with CHX on 58s by Wang et al.(1991).
Zhang et al. (1992) showed that the temperature, at which the fertility of 58s is sensitive to photoperiod, ranged from 24°C (light 26°C/dark 22°C) to 30°C (light 32°C/dark 28°C). In our experiment, the average temperature was within the range mentioned above. Under the same LD condition, the percent of pollen fertility of the control was 0. This shows that the temperature effect could be excluded. Therefore, the partial recovery of pollen fertility under LD sterile condition caused by treatment with CHX was not due to temperature effect. The CHX treatment on 58s lowered pollen fertility in fertile condition while restored pollen fertility partially in sterile condition. It indicated that fertility transformation of 58s is related to the expression of some genes in translation level. Results of 2D patterns of leaf proteins at stage V revealed that the anther was aborted when protein spot [MW59.6kD, pI5.9] existed, and pollen fertility restored partially as it disappeared after treatment with CHX. It is probably the expressed product of photoperiod-sensitive genie male-sterile gene, whose physiological function might be the negative regulation of the expression of fertility. The characteristics of this kind of protein was that the pollen was 100% sterile when they appeared, and partially fertile when they disappeared or did not express. After treatment with CHX, leaves were damaged, pollen fertility was partially restored, but incompletely, so percent of self fertility was still 0. Therefore, the protein could be only one of the regulating factors affecting fertility transformation of PGMR 58s.
The 59.6 kD protein in this paper, is similar to that of 60kD observed by Bai and Tan (1991) and that of 61kD reported by Wang and Tong (1992). They are quite similar in the position of 2D patterns and could be the same protein. This possibility may be verified by techniques such as immuno-precipitation and western-blotting.
Acknowledgment: This study was supported by the grants of China National Natural Science Foundation. We thank Prof. Huang Da-nian of China National Rice Research Institute for the help on 2D electrophoresis.
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