Sirkka Sutinen

Inactivation of the clpC1 Gene Encoding a Chloroplast Hsp100 Molecular Chaperone Causes Growth Retardation, Leaf Chlorosis, Lower Photosynthetic Activity, and a Specific Reduction in Photosystem Content

ClpC is a molecular chaperone of the Hsp100 family. In higher plants there are two chloroplast-localized paralogs (ClpC1 and ClpC2) that are approximately 93% similar in primary sequence. In this study, we have characterized two independent Arabidopsis (Arabidopsis thaliana) clpC1 T-DNA insertion mutants lacking on average 65% of total ClpC content. Both mutants display a retarded-growth phenotype, leaves with a homogenous chlorotic appearance throughout all developmental stages, and more perpendicular secondary influorescences. Photosynthetic performance was also impaired in both knockout lines, with relatively fewer photosystem I and photosystem II complexes, but no changes in ATPase and Rubisco content. However, despite the specific drop in photosystem I and photosystem II content, no changes in leaf cell anatomy or chloroplast ultrastructure were observed in the mutants compared to the wild type. Previously proposed functions for envelope-associated ClpC in chloroplast protein import and degradation of mistargeted precursors were examined and shown not to be significantly impaired in the clpC1 mutants. In the stroma, where the majority of ClpC protein is localized, marked increases of all ClpP paralogs were observed in the clpC1 mutants but less variation for the ClpR paralogs and a corresponding decrease in the other chloroplast-localized Hsp100 protein, ClpD. Increased amounts of other stromal molecular chaperones (Cpn60, Hsp70, and Hsp90) and several RNA-binding proteins were also observed. Our data suggest that overall ClpC as a stromal molecular chaperone plays a vital role in chloroplast function and leaf development and is likely involved in photosystem biogenesis.

Structural and Functional Insights into the Chloroplast ATP-Dependent Clp Protease in Arabidopsis

In contrast with the model Escherichia coli Clp protease, the ATP-dependent Clp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple ClpP and ClpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded ClpP subunit, ClpP6, we show that the Arabidopsis thaliana Clp protease is vital for chloroplast development and function. Repression of ClpP6 produced a proportional decrease in the Clp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the ClpP1 and ClpR1-4 proteins, the other containing ClpP3-6. Proteomic analysis revealed several stromal proteins more abundant in clpP6 antisense lines, suggesting that some are substrates for the Clp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal Clp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the clpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, and quality control, rather than in metabolic activities such as photosynthesis.

Electrical impedance analysis in plant tissues: on the biological meaning of Cole-Cole α in Scots pine needles

Electrical impedance spectra (80 Hz–1 MHz) in Scots pine needles were found to be characterized by spectrum skewness in the Cole-Cole plot. These spectra were subjected to analysis with two distributed models: (i) the Cole-Cole function and (ii) an equivalent circuit which takes account of the presence of air spaces within the needles (Model-A). In analysis with untreated needles (without artificial infiltration with water), Model-A fitted better than the Cole-Cole function to the experimental data. After infiltration of water into the needles, the extent of spectrum skewness was substantially decreased compared with the pre-infiltration condition and the Cole-Cole function fitted better than Model-A to the measured impedance data. The Cole-Cole α decreased from 0.47 in non-infiltrated needles to 0.42 in the infiltrated needles. The exceptionally large value of α in non-infiltrated needles can be explained by the presence of air spaces, which produce transmission line properties in the mesophyll. In support of the validity of Model-A, this new model provided specific membrane resistances of 1190 ± 83 Ω cm2 in cold hardened and non-hardened needles respectively. These specific membrane resistance are comparable with previous reports of membrane resistances in other biological systems. It is concluded that in this exceptionally spongy tissue, Cole-Cole α is likely to be due to the effects of the transmission line properties of cells which are surrounded by air spaces and only thin cell walls outside the insulating cell membranes.

Exposure to chlorinated acetic acids: Responses of peroxidase and glutathione S-transferase activity in pine needles

During long-term exposure of pine (Pinus sylvestris L.) seedlings to trichloro- and monochloroacetic acids via root uptake or acid mist treatments, both substances were removed from the plant tissues by metabolic activity. None of the treated plants exhibited visible stress symptoms at the concentrations used. In addition, the exposure to both substances led to dramatic changes in the activity of xenobiotic detoxification enzymes (peroxidase and gluthatione S-transferase) in the needles of the plants.

Assessment of UV Biological Spectral Weighting Functions for Phenolic Metabolites and Growth Responses in Silver Birch Seedlings

In research concerning stratospheric ozone depletion, action spectra are used as biological spectral weighting functions (BSWFs) for describing the effects of UV radiation on plant responses. Our aim was to evaluate the appropriateness of six frequently used BSWFs that differ in effectiveness with increasing wavelength. The evaluation of action spectra was based on calculating the effective UV radiation doses according to 1–2) two formulations of the generalized plant action spectrum, 3) a spectrum for ultraviolet induced erythema in human skin, 4) a spectrum for the accumulation of a flavonol in Mesembryanthemum crystallinum, 5) a spectrum for DNA damage in alfalfa seedlings and 6) the plant growth action spectrum. We monitored effects of UV radiation on the concentration of individual UV absorbing metabolites and chlorophyll concentrations in leaves and growth responses of silver birch (Betula pendula) seedlings. Experiments were conducted outdoors using plastic films attenuating different parts of the UV spectrum. Chlorophyll concentrations and growth were not affected by the UV treatments. The response to UV radiation varied between and within groups of phenolics. In general, the observed responses of phenolic groups and individual flavonoids were best predicted by action spectra extending into the UV‐A region with moderate effectiveness.

Combined effect of elevated UVB, elevated temperature and fertilization on growth, needle structure and phytochemistry of young Norway spruce (Picea abies) seedlings

Simultaneously with warming climate, other climatic and environmental factors are also changing. Here, we investigated for the first time the effects of elevated temperature, increased ultraviolet-B (UVB) radiation, fertilization and all combinations of these on the growth, secondary chemistry and needle structure of 1-year-old Norway spruce (Picea abies (L.) Karst.) seedlings in an outdoor experiment. After one growing season, elevated temperature increased root : shoot ratio and concentrations of needle piperidine alkaloids, while concentrations of needle catechins and acetophenones and bark flavonoids decreased compared with ambient temperature seedlings. UVB-radiation increased concentrations of bark condensed tannins, while fertilization increased total biomass and concentrations of needle catechins. In addition to the main effects, concentrations of some individual phenolic compounds showed UV × temperature or UV × temperature × fertilization interactions, and fertilization modified temperature response on root : shoot ratio. All the treatments described here affected the defence chemistry profiles of the seedlings, which may imply some changes in plant-herbivore interactions in connection with changing climate. The interactions between treatments indicate a need for further experiments involving several simultaneously affecting environmental changes.

Development and growth of primordial shoots in Norway spruce buds before visible bud burst in relation to time and temperature in the field.

The timing of bud development in ecodormancy is critical for trees in boreal and temperate regions with seasonally alternating climates. The development of vegetative buds and the growth of primordial shoots (the primordial shoot ratio) in Norway spruce were followed by the naked eye and at stereo and light microscopic levels in fresh-cut and fixed buds obtained by regular field samplings during the spring of 2007, 2008 and 2009. Buds were collected from 15 randomly selected trees (all 16 years old in 2007) of one southern Finnish half-sib family. The air temperature was recorded hourly throughout the observation period. In 2008 and 2009, initial events in the buds, seen as accumulation of lipid droplets in the cortex area, started in mid-March and were depleted in late April, simultaneously with the early development of vascular tissue and primordial needles. In mid-April 2007, however, the development of the buds was at least 10 days ahead as a result of warm spells in March and early April. Variation in the timing of different developmental phases within and among the sample trees was negligible. There was no clear one-to-one correspondence between the externally visible and the internal development of the buds. The dependence of the primordial shoot ratio on different types of temperature sum was studied by means of regression analysis. High coefficients of determination (R(2) ≈ 95%) were attained with several combinations of the starting time (beginning of the year/vernal equinox), the threshold value (from -3 to +5 °C), and the time step (hour/day) used in the temperature summation, i.e., the prediction power of the primordial shoot ratio models turned out to be high, but the parameter estimate values were not unambiguous. According to our results, temperature sums describe the growth of the primordial shoot inside the bud before bud burst. Thus, the results provide a realistic interpretation for the present phenological models of bud development that are based on temperature sums and external observations of bud burst only, and they also provide new tools for improving the models.

Visible and microscopic needle alterations of mature Aleppo pine (Pinus halepensis) trees growing on an ozone gradient in eastern Spain.

Visible injuries and 42 microscopic features of tissue and cell structure were quantified in needles of mature Aleppo pine (Pinus halepensis) growing at four field sites located on a natural ozone gradient in eastern Spain. Principal component analysis was used to find out if the forest sites differed from each other, to determine the reasons for the site differences and to evaluate the relations between the parameters studied. In previous-year needles, the first principal component (PC) was described by changes typical of long-term ozone stress: high occurrence of microscopic changes indicating increased defence and faint chlorotic mottling, but low occurrence of ultrastructural changes related to photosynthesis and its storage products. The second PC was described by needle ageing or ontological senescence. Statistical differences between the sites in terms of ozone stress were found and were in line with measured ozone concentrations and the values of the ozone exposure index, AOT40. Symptoms of ozone stress were mild, i.e., not related to severe tissue damage. Results suggested that the faint chlorotic mottling can be attributed to certain forms of condensed tannins or small chloroplasts. In addition, a coastal site differed from mountainous sites by having a more mesomorphic needle anatomy.

Implications of delayed soil thawing on trees: A case study of a Picea abies stand

Abstract The effects of experimentally delayed soil thawing were studied on a 32-year-old stand of Norway spruce [Picea abies (L.) Karst.] located in the eastern part of Finland. While the soil temperature at depths of 10 and 50 cm rose above 0°C at the beginning of May in the control treatment (CTRL), it stayed close to 0°C until mid-July in the delayed soil-thawing treatment (FROST). Budburst was delayed by a few days by the FROST treatment. Treatment affected the physiology of previous- and current-year needles, i.e. their electrical impedance, potential efficiency of photosystem II (F v/F m), and chlorophyll a and b content. At the end of the growing season, in November, both the area of the central cylinder and all of the measured dimensions inside the cylinder in the current needles and number of sieve cells in the previous years needles were smaller in the FROST than the CTRL trees. At the same time, the fully developed dormant buds showed differences only in their side shoots, the width of the buds being smaller in the FROST than the CTRL trees. No effect was found on the onset of wood formation. In contrast, the radial increment tended to be faster in the delayed soil-thawing treatment during late summer.

Physiological changes in Pinus sylvestris needles during early spring under sub-arctic conditions.

Abstract Physiological condition of yellow and visually green Scots pine (Pinus sylvestris L.) needles was followed in the Finnish sub-arctic during May and June 1996. The five greenest and five yellowest saplings were chosen for needle sampling at weekly intervals. The chlorophyll fluorescence, chlorophyll and water content, fine structure of the mesophyll cells and frost hardiness were determined at weekly intervals. Intracellular, extracellular and cell-membrane resistance were estimated by impedance spectroscopy and the distribution of free and bound water were determined by magnetic resonance imaging for needle samples collected on June 11, 1996. The chlorophyll a and b contents, the ratio between the maximum variable fluorescence and the maximal fluorescence yield (Fv/Fm) and the thickness of the grana stacks were higher in the green than yellow needles during the first three samplings in May. The yellow needles maintained a higher level of freezing-stress resistance compared to the green needles. The recovery of green color was accompanied by an increase in the chlorophyll a and b content, Fv/Fm and the thickening of the grana stacks. The water content was slightly higher in the green than yellow needles in June. The water content still decreased even though the chlorophyll content of yellow needles began to increase and approached that of the green needles. Based on the magnetic resonance imaging, the water was distributed differently and in a more mobile form in the yellow than in the green needles. The extracellular resistance and the cell-membrane resistance were lower in yellow than green needles indicating impaired ability of yellow needles to maintain a high intracellular ion concentration. The results show that the yellow color of needles is an indication of a deeper state of photo-inhibition and slower deacclimation and is not directly related to the desiccation stress.