Elucidating the molecular details of qE (energy quenching) induction in higher plants has proven to be a major challenge. Identification of qE mutants has provided initial information on functional ...elements involved in the qE mechanism; furthermore, investigations on isolated pigment-protein complexes and analysis in vivo and in vitro by sophisticated spectroscopic methods have been used for the elucidation of mechanisms involved. The aim of the present review is to summarize the current knowledge of the phenotype of npq (non-photochemical quenching)-knockout mutants, the role of gene products involved in the qE process and compare the molecular models proposed for this process.
Main conclusion
Simultaneous genome editing of the two homeologous
LCYe
and
ZEP
genes of
Nicotiana benthamiana
results in plants in which all xanthophylls are replaced by zeaxanthin
.
Plant ...carotenoids act both as photoreceptors and photoprotectants in photosynthesis and as precursors of apocarotenoids, which include signaling molecules such as abscisic acid (ABA). As dietary components, the xanthophylls lutein and zeaxanthin have photoprotective functions in the human macula. We developed transient and stable combinatorial genome editing methods, followed by direct LC–MS screening for zeaxanthin accumulation, for the simultaneous genome editing of the two homeologous
Lycopene Epsilon Cyclase
(
LCYe
) and the two
Zeaxanthin Epoxidase
(
ZEP
) genes present in the allopolyploid
Nicotiana benthamiana
genome. Editing of the four genes resulted in plants in which all leaf xanthophylls were substituted by zeaxanthin, but with different ABA levels and growth habits, depending on the severity of the
ZEP1
mutation. In high-zeaxanthin lines, the abundance of the major photosystem II antenna LHCII was reduced with respect to wild-type plants and the LHCII trimeric state became unstable upon thylakoid solubilization. Consistent with the depletion in LHCII, edited plants underwent a compensatory increase in PSII/PSI ratios and a loss of the large-size PSII supercomplexes, while the level of PSI-LHCI supercomplex was unaffected. Reduced activity of the photoprotective mechanism NPQ was shown in high-zeaxanthin plants, while PSII photoinhibition was similar for all genotypes upon exposure to excess light, consistent with the antioxidant and photoprotective role of zeaxanthin in vivo.
In Chlamydomonas reinhardtii, the model organism for eukaryotic green algae and plants, the processes of nuclear transformation and genome editing in particular are still marked by a low level of ...efficiency, and so intensive work is required in order to create and identify mutants for the investigation of basic physiological processes, as well as the implementation of biotechnological applications. In this work, we show that cell synchronization during the stages of the cell cycle, obtained from long-term cultivation under specific growth conditions, greatly enhances the efficiency of transformation and allows the identification of DNA repair mechanisms that occur preferentially at different stages of the cell cycle. We demonstrate that the transformation of synchronized cells at different times was differentially associated with nonhomologous end joining (NHEJ) and/or homologous recombination (HR), and makes it possible to knock-in specific foreign DNA at the genomic nuclear location desired by exploiting HR. This optimization greatly reduces the overall complexity of the genome editing procedure and creates new opportunities for altering genes and their products.
Display omitted
•Deletion of PSI antenna unbalances photosynthesis under intermittent light conditions.•A complete PSI supercomplex endowed with red-spectral forms optimizes the thylakoid electron ...flow.•Plants evolved antenna systems with distinct spectral properties for PSI vs. PSII.•Such a diversity likely contributed to maintaining the PQ redox state and avoiding photoinhibition under variable lighting.
Higher plant Photosystem I (PSI) includes a peripheral antenna system (LHCI) composed of four light-harvesting proteins (Lhca1-Lhca4). The LHCI system is highly conserved, suggesting that it plays a specific role within the LHC family. In order to elucidate the specific function of LHCI, the phenotype of an Arabidopsis mutant devoid of the whole LHCI system was studied over a range of conditions, including rapid changes in irradiation. ΔLhca plants displayed stunted growth and reduced thylakoid lumen acidification respect to wild type, suggesting that the lack of LHCI affected electron transport rate. Under rapidly changing light intensity, growth rate of the mutant was further reduced and the redox balance of the photosynthetic electron chain impaired. Instead, under constant, excess light regime, the ΔLhca plants did not suffer enhanced photooxidation vs. wild type, implying LHCI optimizes the flow rate through the electron transport chain by maintaining high PSI activity at all irradiances. We conclude that a complete PSI supercomplex, including LHCI, is crucial for the dynamic regulation of PQ redox state and therefore for PSII photoprotection.
Non-photochemical quenching is the photoprotective heat dissipation of chlorophyll-excited states. In higher plants, two quenching sites are located in trimeric LHCII and monomeric CP29 proteins. ...Catalysis of dissipative reactions requires interactions between chromophores, either carotenoid, chlorophyll or both. We identified CP29 protein domains involved in quenching by complementing an Arabidopsis deletion mutant with sequences deleted in pigment-binding or pH-sensitive sites. Acidic residues exposed to the thylakoid lumen were found not essential for activation of thermal dissipation in vivo. Chlorophylls a603 (a5) and a616 were identified as components of the catalytic pigment cluster responsible for quenching reaction(s), in addition to xanthophyll L2 and chlorophyll a609 (b5). We suggest that a conformational change induced by acidification in PsbS is transduced to CP29, thus bringing chlorophylls a603, a609 and a616 into close contact and activating a dissipative channel. Consistently, mutations on putative protonatable residues, exposed to the thylakoid lumen and previously suggested to regulate xanthophyll exchange at binding site L2, did not affect quenching efficiency.
The induction, i.e., the rapid increase of nitrate (
NO
3
–
) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses ...to
NO
3
–
treatment were characterized in terms of changes in
NO
3
–
uptake rate and plasma membrane (PM) H
+
-ATPase activity and related to transcriptional and protein profiles of
NRT2, NRT3
, and
PM H
+
-ATPase
gene families. The behavior of transcripts and proteins of
ZmNRT2s
and
ZmNRT3s
suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified
PM H
+
-ATPase
transcripts indicates that those mainly involved in the response to
NO
3
–
treatment are
ZmHA2
and
ZmHA4
. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H
+
-ATPase can occur
in vivo
as hexameric complex together with the already described monomeric and dimeric forms.
Natural light harvesting is exceptionally efficient thanks to the local energy funnel created within light-harvesting complexes (LHCs). To understand the design principles underlying energy transport ...in LHCs, ultrafast spectroscopy is often complemented by mutational studies that introduce perturbations into the excitonic structure of the natural complexes. However, such studies may fall short of identifying all excitation energy transfer (EET) pathways and their changes upon mutation. Here, we show that a synergistic combination of first-principles calculations and ultrafast spectroscopy can give unprecedented insight into the EET pathways occurring within LHCs. We measured the transient absorption spectra of the minor CP29 complex of plants and of two mutants, systematically mapping the kinetic components seen in experiments to the simulated exciton dynamics. With our combined strategy, we show that EET in CP29 is surprisingly robust to the changes in the exciton states induced by mutations, explaining the versatility of plant LHCs.
We combine site-directed mutagenesis with picosecond time-resolved fluorescence and femtosecond transient absorption (TA) spectroscopies to identify excitation energy transfer (EET) processes between ...chlorophylls (Chls) and xanthophylls (Xant) in the minor antenna complex CP29 assembled inside nanodiscs, which result in quenching. When compared to WT CP29, a longer lifetime was observed in the A2 mutant, missing Chl a612, which closely interacts with Xant Lutein in site L1. Conversely, a shorter lifetime was obtained in the A5 mutant, in which the interaction between Chl a603 and Chl a609 is strengthened, shifting absorption to lower energy and enhancing Chl-Xant EET. Global analysis of TA data indicated that EET from Chl a Q y to a Car dark state S* is active in both the A2 and A5 mutants and that their rate constants are modulated by mutations. Our study provides experimental evidence that multiple Chl–Xant interactions are involved in the quenching activity of CP29.
Proper assembly of plant photosystem II, in the appressed region of thylakoids, allows for both efficient light harvesting and the dissipation of excitation energy absorbed in excess. The core moiety ...of wild type supercomplex is associated with monomeric antennae that, in turn, bind peripheral trimeric LHCII complexes. Acclimation to light environment dynamics involves structural plasticity within PSII-LHCs supercomplexes, including depletion in LHCII and CP24. Here, we report on the acclimation of NoM, an Arabidopsis mutant lacking monomeric LHCs but retaining LHCII trimer. Lack of monomeric LHCs impaired the operation of both photosynthetic electron transport and state transitions, despite the fact that NoM underwent a compensatory over-accumulation of the LHCII complement compared to the wild type. Mutant plants displayed stunted growth compared to the wild type when probed over a range of light conditions. When exposed to short-term excess light, NoM showed higher photosensitivity and enhanced singlet oxygen release than the wild type, whereas long-term acclimation under stress conditions was unaffected. Analysis of pigment-binding supercomplexes showed that the absence of monomeric LHCs did affect the macro-organisation of photosystems: large PSI-LHCII megacomplexes were more abundant in NoM, whereas the assembly of PSII-LHCs supercomplexes was impaired. Observation by electron microscopy (EM) and image analysis of thylakoids highlighted impaired granal stacking and membrane organisation, with a heterogeneous distribution of PSII and LHCII compared to the wild type. It is concluded that monomeric LHCs are critical for the structural and functional optimisation of the photosynthetic apparatus.
•Peripheral antennae allow for both efficient light harvesting and the dissipation of excess energy.•Deletion of minor antennae impaired the operation of photosynthetic electron transport.•In mutant plants, clusters of PSII cores and LHCII-only domains coexist in a disorderly manner.•The results guide the engineering strategy to optimise antenna size in crops species.
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