Grzegorz Jackowski

Identification of Lhcb1/Lhcb2/Lhcb3 heterotrimers of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II).

Using non-denaturing isoelectric focusing in polyacrylamide vertical slab gel, we have purified to homogeneity three trimeric subcomplexes of LHC II from Arabidopsis thylakoid membranes. The polypeptide composition of the subcomplexes were studied by immunoblotting. Our results indicate the existence in vivo of LHC II heterotrimers containing Lhcb1, Lhcb2 and Lhcb3 gene products.

The thylakoid protease Deg2 is involved in stress-related degradation of the photosystem II light-harvesting protein Lhcb6 in Arabidopsis thaliana

• The thylakoid protease Deg2 is a serine-type protease peripherally attached to the stromal side of the thylakoid membrane. Given the lack of knowledge concerning its function, two T-DNA insertion lines devoid of Deg2 were prepared to study the functional importance of this protease in Arabidopsis thaliana. • The phenotypic appearance of deg2 mutants was studied using a combination of stereo and transmission electron microscopy, and short-stress-mediated degradation of apoproteins of minor light-harvesting antennae of photosystem II (PSII) was analysed by immunoblotting in the mutants in comparison with wild-type plants. • Deg2 repression produced a phenotype in which reduced leaf area and modified chloroplast ultrastructure of older leaves were the most prominent features. In contrast to the wild type, the chloroplasts of second-whorl leaves of 4-wk-old deg2 mutants did not display features typical of the early senescence phase, such as undulation of the chloroplast envelope and thylakoids. The ability to degrade the photosystem II light-harvesting protein Lhcb6 apoprotein in response to brief high-salt, wounding, high-temperature and high-irradiance stress was demonstrated to be impaired in deg2 mutants. • Our results suggest that Deg2 is required for normal plant development, including the chloroplast life cycle, and has an important function in the degradation of Lhcb6 in response to short-duration stresses.

The acclimative response of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) to elevated irradiances at the level of trimeric subunits.

The changes in structural organization of the major light-harvesting chlorophyll a/b-protein complex of photosystem II (LHC II) at the level of trimeric subcomplexes were studied in spinach plants grown under low light conditions (50 micromol quanta m(-2) s(-1)) and then acclimated to elevated irradiances. By monitoring photochemical quenching of fluorescence yield (qP), photosystem II (PS II) functional status was assessed in leaves of plants acclimated to a range of elevated irradiances. Three separate acclimative irradiances were selected for the experiments, reflecting: limiting light conditions (150 micromol quanta m(-2) s(-1)), near to the inflexion point on the irradiance curve conditions (300 micromol quanta m(-2) s(-1)) and an excessive light, causing a moderate stress in the form of down regulation of PS II (450 micromol quanta m(-2) s(-1)). An immunoblot analysis showed that there was a clear decline in an abundance on chlorophyll basis of Lhcb1-3 apoproteins as an acclimative irradiance increased from 50 to 450 micromol quanta m(-2) s(-1), with Lhcb1 decreasing to a lesser extent than Lhcb2 and Lhcb3 (only at excessive irradiance). When analyzed by non-denaturing isoelectric focusing BBY membrane fragments (PSIIalpha-enriched, stacked thylakoid membranes) isolated from low light-grown plants were resolved into nine fractions, seven of which (labelled 3-9) were established by us previously [Jackowski and Pielucha, J. Photochem. Photobiol. B: Biol. 64 (2001) 45] to be LHC II subcomplexes representing mixed populations of closely similar trimers, comprising permutations of Lhcb1 and Lhcb2 (subcomplexes 3-7) or Lhcb1-3 (subcomplexes 8 and 9). A heterogeneity with regard to accumulation behaviour of LHC II subcomplexes in response to elevated irradiances was revealed. The subcomplexes 5 and 6 were accumulating at similar level, regardless of the light irradiance experienced. Another group consisting of the subcomplexes 3 and 4 (the most basic ones) showed a progressive increase in relative abundance with increasing an irradiance intensity whereas the subcomplexes 7-9 (the most acidic ones) exhibited a progressive decline in their relative abundance during an acclimation of spinach plants to elevated irradiances thus they may collectively represent an elevated irradiance-responsive subunit of LHCII.

Characterization of Photosystem II Antenna Complexes Separated by Non-Denaturing Isoelectric Focusing

The light-harvesting chlorophyll a/b-binding protein complexes of photosystem II (PSII) are a family of pigment-proteins accounting for the majority of PSII chlorophyll (chi) and protein in the thylakoid membrane. They bind chl a and b and the xanthophylls lutein, violaxanthin, neoxanthin, antheraxanthin and zeaxanthin. The main chl a/bprotein complex of PSII — LHCII — acts primarily as light-harvesting antenna but was found to be involved also in thylakoid adhesion and the acclimation of photosynthetic apparatus to short -term and prolonged changes in the light regime. Besides LHCII at least three minor chl a/b-proteins are found in PSII particles, i.e. CP29, CP26 and CP24. These complexes function as intermediates in transfer of excitation energy form LHCII to PSII core although they may also serve as sites of thermal deactivation of excess excitation energy within PSII.

Involvement of Deg5 protease in wounding-related disposal of PsbF apoprotein.

Deg5 is a serine-type protease peripherally attached to luminal side of thylakoid membrane. Given the lack of knowledge concerning its function homozygous T-DNA insertion line depleted in Deg5 was prepared to study the functional importance of this protease in Arabidopsis thaliana. deg5 mutants displayed a pleiotropic phenotype with regard to fourth whorl leaves of four-weeks old plants. The alterations involved an increased leaf area, reduced leaf thickness, reduced cross-sectional area of palisade mesophyll cells as well as changed chloroplast ultrastructure including lack of signs of entering the senescence phase (e.g. presence of much smaller plastoglobules) and the accumulation of large starch grains at the end of the dark period. It was shown that whereas PsbA, C and F apoproteins of photosystem II reaction center undergo an extensive disappearance in response to a set of brief stresses deg5 mutant was fully resistant to the disappearance of PsbF apoprotein which follows an exposition of leaves to wounding. Our results demonstrate that Deg5 is of seminal importance for normal plant development and degradation of PsbF which occurs following brief wounding.

The resolution and biochemical characterization of subcomplexes of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II)

LHC II isolated from carnation leaves has been solubilized and resolved by a newly developed, vertical-bed non-denaturing isoelectric focusing in polyacrylamide slab gels to yield three trimeric subcomplexes focusing at pH 4.52, 4.42 and 4.37 (designated a, b and c, respectively), comprising approximately 38%, 24% and 38% of the chlorophyll. The spectroscopic data demonstrated a close similarity among LHC II subcomplexes concerning their chlorophyll content and organization. The most alkaline and the most acidic subcomplex contained the 27 kDa polypeptide of LHC II while the intermediate pI fraction contained both LHC II polypeptides, i.e. 27 kDa and 26 kDa ones associated at 2:1 stoichiometry. The 27 kDa polypeptide could be resolved by denaturing isoelectrofocusing into 10 pI molecular isoforms covering 5.90–4.20 pH range. Three of the isoforms were found in the subcomplexes a and b and eight in the subcomplex c. The 26 kDa polypeptide comprised the unique pI molecular isoform focusing at pH 5.61.

AtFtsH heterocomplex-mediated degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to stresses

Chloroplastic heterocomplex consisting of AtFtsH1, 2, 5 and 8 proteases, integrally bound to thylakoid membrane was shown to play a critical role in degradation of photodamaged PsbA molecules, inherent to photosystem II (PSII) repair cycle and in plastid development. As no one thylakoid bound apoproteins besides PsbA has been identified as target for the heterocomplex-mediated degradation we investigated the significance of this protease complex in degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to various stressing conditions and in stress-related changes in overall composition of LHCII trimers of PSII-enriched membranes (BBY particles). To reach this goal a combination of approaches was applied based on immunoblotting, in vitro degradation and non-denaturing isoelectrofocusing. Exposure of Arabidopsis thaliana leaves to desiccation, cold and high irradiance led to a step-wise disappearance of Lhcb1 and Lhcb2, while Lhcb3 level remained unchanged, except for high irradiance which caused significant Lhcb3 decrease. Furthermore, it was demonstrated that stress-dependent disappearance of Lhcb1-3 is a proteolytic phenomenon for which a metalloprotease is responsible. No changes in Lhcb1-3 level were observed due to exposition of var1-1 mutant leaves to the three stresses clearly pointing to the involvement of AtFtsH heterocomplex in the desiccation, cold and high irradiance-dependent degradation of Lhcb1 and Lhcb2 and in high irradiance-dependent degradation of Lhcb3. Non-denaturing isoelectrofocusing analyses revealed that AtFtsH heterocomplex-dependent differential Lhcb1-3 disappearance behaviour following desiccation stress was accompanied by modulations in abundances of individual LHCII trimers of BBY particles and that LHCII of var1-1 resisted the modulations.

Lhca polypeptides are maintained during dark-induced senescence of leaves

Thylakoid polypeptides samples from fresh and dark-incubated (0–6 days) leaves were tested for changes in relative abundancies of Lhca polypeptides using a set of polyclonal and monoclonal antibodies in conjunction with immunoblot technique and integrating densitometry. All Lhca polypeptides were found to be more stable within 6 days of dark incubation than bulk chlorophyll itself. On chlorophyll basis the relative abundance of Lhca 1–4 increased after 6 days of dark incubation to 154, 177, 144 and 121 %, respectively, of the values found in fresh leaves.

Kinetin modifies the secondary structure of poly(A)RNA in cucumber cotyledons

Abstract Evidence has been obtained for a presence of double-stranded regions in poly(A)RNA from cucumber cotyledons, as well as for an engagement of the poly(A) segment in the formation of these regions. Kinetic treatment of the cotyledons leads to an increase in degree of the secindary structure of poly(A)RNA, to a slight increase in the length of the poly(A) tail and to a considerable decrease in its involvement in base pairing, thus making the poly(A) segments more ‘unmasked’.

The irradiance dependent transcriptional regulation of AtCLPB3 expression.

Transcript abundance analysis was applied to determine whether expression of genes coding for 50 principal constituents of chloroplast and mitochondria proteolytic machinery, i.e. isoforms of proteases and regulatory subunits of Clp and FtsH families as well as Deg group of chymotrypsin family are differentially expressed in response to acclimation to elevated irradiance. Of 50 genes analysed only a single one coding for ClpB3 regulatory subunit was found to be up-regulated and gene coding for Deg2 to be down-regulated significantly during acclimation to excessive irradiance conditions. Hierarchical clustering of transcript abundance data revealed that CLPB3 co-expressed tightly with genes coding for PAP1, GBF6 and bHLH family member transcription factors during the acclimation. It was found that CLPB3 contains cis-regulatory elements able to bind all three transcription factors. By performing analyses of publicly available transcriptomic data sets from a range of long-term abiotic stresses we suggest that PAP1 may mediate condition-dependent transcriptional response of CLPB3, induced by a group of long-term abiotic stresses.