Identification and differential accumulation of two isoforms of the CF1-beta subunit under high light stress in Brassica rapa.

The chloroplast ATP synthase coupling factor CF1 complex contains five nonidentical subunits, alpha, beta, gamma, delta, and epsilon, with a stoichiometry of 3:3:1:1:1. The beta subunit contains the catalytic site for ATP synthesis during photooxidative phosphorylation in the chloroplast. In this study, we have identified two isoforms of the CF1-beta subunit at 56 and 54 kDa in the chloroplast of Brassica rapa, through isolation/purification, proteolytic digestion and internal peptide sequencing. Examining their accumulation pattern demonstrates that both isoforms coexist during chloroplast biogenesis and in mature thylakoid membranes, but the 54 kDa isoform is more apparently upregulated by light or under light stress. LiDS-PAGE shows that the 56 kDa is a major isoform of the CF1-beta subunit under normal light conditions, and its amount was not influenced during high light or other light stress treatments. The 54 kDa isoform is a minor band at normal conditions; however, it significantly increased under excess light stresses, such as high or low light with drought and/or high temperature. Particularly, a ninefold increase was observed after 8-10 h of high light treatment with drought and high temperature. The results suggest that light stress induction of the 54 kDa CF1-beta isoform may present a positive response during chloroplast photoacclimation.

Brassica rapa plants adapted to microgravity with reduced photosystem I and its photochemical activity.

The photosynthetic apparatus contains several protein complexes, many of which are regulated by environmental conditions. In this study, the influences of microgravity on PSI and PSII in Brassica rapa plants grown aboard the space shuttle were examined. We found that Brassica plants grown in space had a normal level of growth relative to controls under similar conditions on Earth. Upon return to Earth, cotyledons were harvested and thylakoid membranes were isolated. Analysis of chlorophyll contents showed that the Chl a/b ratio (3.5) in flight cotyledons was much higher than a ratio of 2.42 in the ground controls. The flight samples also had a reduction of PSI complexes and a corresponding 30% decrease of PSI photochemical activity. Immunoblotting showed that the reaction centre polypeptides of PSI were more apparently decreased (e.g. by 24-33% for PsaA and PsaB, and 57% for PsaC) than the light-harvesting complexes. In comparison, the accumulation of PSII complex was less affected in microgravity, thus only a slight reduction in D1, D2 and LHCII was observed in protein blots. However, there was a 32% decrease of OEC1 in the flight samples, indicating a defective OEC subcomplex. In addition, an average 54% increase of the 54 kDa CF1-beta isoform was found in the flight samples, suggesting that space-grown plants suffered from certain stresses, consistent with implications of the increased Chl a/b ratio. Taken together, the results demonstrated that Brassica plants can adapt to spaceflight microgravity, but with significant alterations in chloroplast structures and photosynthetic complexes, and especially reduction of PSI and its activity.

Rice phospholipase D isoforms show differential cellular location and gene induction.

Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, stress responses, protein trafficking, and membrane metabolism. This report describes the cloning and characterization of three novel PLD genes from rice, designated RPLD3, RPLD4 and RPLD5. The rice PLDs, including the previously isolated RPLD1 and RPLD2, are similar to PLD subfamilies of Arabidopsis: Based on sequence homology and domain conservation, RPLD1 is most similar to the PLDalpha subfamily of PLDs while RPLD5 most closely resembles the PLDdelta type. RPLD2, 3 and 4 represent a unique subfamily, although they are most similar to PLDalpha. RPLD1 is located on chromosome 1, RPLD5 on chromosome 3, and RPLD2, RPLD3, and RPLD4 are tandemly arrayed on chromosome 5. Transcriptional analysis reveals that RPLD1, present in healthy rice vegetative tissues, is induced rapidly but transiently in wounded leaf tissues. RPLD2, also induced by wounding, is present at lower levels but for a more prolonged duration than RPLD1. Immunolocalization with peptide specific antibodies to each of the five PLDs was used to demonstrate that the isoforms have overlapping but distinct patterns of distribution in healthy rice cells. RPLD1 was detected in mesophyll cell wall, membranes, and chloroplasts, whereas RPLD3 and RPLD4 were located predominantly in the chloroplasts. Labeling of RPLD2 and RPLD5 was sparse, and was most concentrated in the secondary walls of xylem (RPLD2) and guard cells (RPLD2 and RPLD5). This combined information on structural features, expression profiles, and cellular localization will assist the basis for dissection of PLD isoform function in rice.