Multi-colour fluorescence imaging of photosynthetic activity and plant stress

Imaging the four fluorescence bands of leaves, the red (F$_{690}$) and far-red (F$_{740}$) chlorophyll (Chl) fluorescence as well as the blue (F$_{440}$) and green (F$_{520}$) fluorescence of leaves and the corresponding fluorescence ratios is a fast and excellent nondestructive technique to detect the photosynthetic activity and capacity of leaves, of gradients over the leaf area as well as the effect of various strain and stress parameters on plants. This review primarily deals with the first and pioneering multi-colour fluorescence imaging results obtained since the mid-1990s in a cooperation with French colleagues in Strasbourg and in my laboratory in Karlsruhe. Together we introduced not only the joint imaging of the red and far-red Chl fluorescence but also of the blue and green fluorescence of leaves. The two instrumental setups composed for this purpose were (1) the Karlsruhe–Strasbourg UV-Laser Fluorescence Imaging System (Laser-FIS) and (2) the Karlsruhe Flash-Light Fluorescence Imaging System (FL-FIS). Essential results obtained with these instruments are summarized as well as the basic principles and characteristics of multi-colour fluorescence imaging. The great advantage of fluorescence imaging is that the fluorescence yield in the four fluorescence bands is sensed of several thousand up to 200,000 pixels per leaf area in one image. The multi-colour FIS technique allows to sense many physiological parameters and stress effects in plants at an early stage before a damage of leaves is visually detectable. Various examples of plant stress detection by the multi-colour FIS technique are given. Via imaging the Chl fluorescence ratio F690/F740 it is even possible to determine the Chl content of leaves. The FIS technique also allows to follow the successive uptake of diuron and loss of photosynthetic function and to screen the ripening of apples during storage. Particularly meaningful and of high statistical relevance are the fluorescence ratio images red/far-red (F$_{690}$/ F$_{740}$), blue/red (F$_{440}$/F$_{690}$), and blue/green (F$_{440}$/F$_{520}$) as well as images of the fluorescence decrease ratio R$_{Fd}$, which is an indicator of the net CO$_{2}$ assimilation rates of leaves

My contact and cooperation with Govindjee over the last five decades: Chlorophyll fluorescence and Rebeiz Foundation

Govindjee and Hartmut Lichtenthaler have a very similar curriculum vitae. Both chose photosynthesis as research field and actively applied chlorophyll fluorescence. Their research was overlapping and complementary. On the occasion of Govindjee\u27s 88th anniversary in 2020, Hartmut Lichtenthaler gives a short retrospective on interactions and joint activities with Govindjee over the past five decades

Contents of photosynthetic pigments and ratios of chlorophyll a/b and chlorophylls to carotenoids (a+b)/(x+c) in C4_{4} plants as compared to C3_{3} plants

The content of chlorophylls (Chl) (a+b), total carotenoids (x+c), and the pigment ratios of Chl a/b and Chls to carotenoids (a+b)/(x+c) of green leaves of five C$_{4}$ plants were determined and compared to those of C$_{3}$ plants. The C$_{4}$ plants were: Pacific and Chinese silvergrass (Miscanthus floridulus and Miscanthus sinensis), sugar cane (Saccharum officinarum) as well as feed and sugar maize (Zea mays). The three C$_{3}$ plants were beech, ginkgo, and oak. C$_{4}$ plants possess higher values for the ratio Chl a/b (3.4-4.5) as compared to the C$_{3}$ plants (2.6-3.3). Sugar maize had the highest values for Chl a/b (4.04-4.70) and exceptionally high contents of total carotenoids and consequently lower values for the ratio of (a+b)/(x+c) (mean: 3.75 ± 0.6). During autumnal senescence also C$_{4}$ plants showed a faster decline of Chl b as compared to Chl a yielding high values for Chl a/b of 6 to 8. Chlorophylls declined faster than carotenoids yielding low (a+b)/(x+c) values below 1.0

Strong increase of photosynthetic pigments and leaf size in a pruned Ginkgo biloba tree

A 50-year-old solitary, sun-exposed ginkgo tree had strongly been pruned in the fall of 2021. Very few buds for the formation of new leaves, twigs, and branches were left over. In spring 2022, these few remaining buds responded with the formation of a different leaf type. These leaves were 2.7 times larger and also thicker than in the years before. In addition, the mean content of total chlorophylls [Chl (a+b)] per leaf area unit of dark-green leaves was 1.45, those of green leaves two times higher as compared to the years leaves as compared to 435 to 770 mg m-2 in leaves of other trees. The higher values for Chl (a+b) and total carotenoid content showed up also on a fresh and dry mass basis. Thus, with the formation of a new, larger leaf type by changes in morphology (leaf size and thickness) and the increase of photosynthetic pigments, the pruned ginkgo tree was able to compensate for the much lower number of leaves and photosynthetic units

Neutron Transfer reactions induced by 8Li on 9Be

Angular distributions for the elastic scattering of 8Li on 9Be and the neutron transfer reactions 9Be(8Li,7Li)10Be and 9Be(8Li,9Li)8Be have been measured with a 27 MeV 8Li radioactive nuclear beam. Spectroscopic factors for 8Li|n=9Li and 7Li|n=8Li bound systems were obtained from the comparison between the experimental differential cross section and finite-range DWBA calculations with the code FRESCO. The spectroscopic factors obtained are compared to shell model calculations and to other experimental values from (d,p) reactions. Using the present values for the spectroscopic factor, cross sections for the direct neutron-capture reactions 7Li(n,g)8Li and 8Li(n,g)9Li were calculated in the framework of a potential model.Comment: 24 pages, 8 Figures, submitted as regular article to PR

Excitated state properties of 20-chloro-chlorophyll a

The excited-state and lasing properties of 20-chloro-chlorophyll a in ether solution were compared to those of chlorophyll a. Desactivation parameters and cross-sections were obtained from non-linear absorption spectroscopy in combination with a physico-mathematical methods package. The Cl substituent at C-20 (1) increases both intersystem crossing and internal conversion, (2) produces a blue-shift of the S1 absorption spectrum, and (3) leads to pronounced photochemistry

Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and γ-Secretase

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development

Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons

Background: The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer’s disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 (SEZ6) and its homolog SEZ6L. Methods and results: We generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins (sSEZ6, sSEZ6L) were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid (CSF) and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice. Conclusions: This study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice

Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons

Background: The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer’s disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 (SEZ6) and its homolog SEZ6L. Methods and results: We generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins (sSEZ6, sSEZ6L) were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid (CSF) and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice. Conclusions: This study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice