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Summary: CRISPR‐Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation.CRISPR‐Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base‐editing systems to induce precise cytosine or adenine conversions in P. patens.Using cytosine or adenine base editors, site‐specific single‐base mutations can be achieved with an efficiency up to 55%, without off‐target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino‐acid changes. Finally, we set up a co‐editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site‐specific base editing in P. patens.These two base editors will facilitate gene functional analysis in P. patens, allowing for site‐specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing. [ABSTRACT FROM AUTHOR]

Summary: Photosynthetic electron transport is regulated by cyclic and pseudocyclic electron flow (CEF and PCEF) to maintain the balance between light availability and metabolic demands. CEF transfers electrons from photosystem I to the plastoquinone pool with two mechanisms, dependent either on PGR5/PGRL1 or on the type I NADH dehydrogenase‐like (NDH) complex. PCEF uses electrons from photosystem I to reduce oxygen and in many groups of photosynthetic organisms, but remarkably not in angiosperms, it is catalyzed by flavodiiron proteins (FLVs).In this study, Physcomitrella patens plants depleted in PGRL1, NDH and FLVs in different combinations were generated and characterized, showing that all these mechanisms are active in this moss.Surprisingly, in contrast to flowering plants, Physcomitrella patens can cope with the simultaneous inactivation of PGR5‐ and NDH‐dependent CEF but, when FLVs are also depleted, plants show strong growth reduction and photosynthetic activity is drastically reduced.The results demonstrate that mechanisms for modulation of photosynthetic electron transport have large functional overlap but are together indispensable to protect photosystem I from damage and they are an essential component for photosynthesis in any light regime. See also the Commentary on this article by Leister, "Leister, 228: 1166–1168. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms support cell metabolism by harvesting sunlight and driving the electron transport chain at the level of thylakoid membranes. Excitation energy and electron flow in the photosynthetic apparatus is continuously modulated in response to dynamic environmental conditions. Alternative electron flow around photosystem I plays a seminal role in this regulation contributing to photoprotection by mitigating overreduction of the electron carriers. Different pathways of alternative electron flow coexist in the moss Physcomitrella patens, including cyclic electron flow mediated by the PGRL1/PGR5 complex and pseudo‐cyclic electron flow mediated by the flavodiiron proteins FLV. In this work, we generated P. patens plants carrying both pgrl1 and flva knock‐out mutations. A comparative analysis of the WT, pgrl1, flva, and pgrl1 flva lines suggests that cyclic and pseudo‐cyclic processes have a synergic role in the regulation of photosynthetic electron transport. However, although both contribute to photosystem I protection from overreduction by modulating electron flow following changes in environmental conditions, FLV activity is particularly relevant in the first seconds after a light change whereas PGRL1 has a major role upon sustained strong illumination. Photosynthesis is continuously modulated in response to dynamic environmental conditions also through modulation of electron transport. In this work, we applied a multidisciplinary approach to demonstrate the fundamental role of mechanisms for regulation of photosynthetic electron transport in plant response to light dynamics. In their absence, photosynthetic apparatus is damaged with strong negative consequences on growth. [ABSTRACT FROM AUTHOR]

ContentsSummary105I.Introduction105II.Diversity of molecular mechanisms for regulation of photosynthetic electron transport106III.Role of FLVs in the regulation of photosynthesis in eukaryotes107IV.Why were FLVs lost in angiosperms?108V.Conclusions108Acknowledgements109References109 Summary: Photosynthetic electron transport requires continuous modulation to maintain the balance between light availability and metabolic demands. Multiple mechanisms for the regulation of electron transport have been identified and are unevenly distributed among photosynthetic organisms. Flavodiiron proteins (FLVs) influence photosynthetic electron transport by accepting electrons downstream of photosystem I to reduce oxygen to water. FLV activity has been demonstrated in cyanobacteria, green algae and mosses to be important in avoiding photosystem I overreduction upon changes in light intensity. FLV‐encoding sequences were nevertheless lost during evolution by angiosperms, suggesting that these plants increased the efficiency of other mechanisms capable of accepting electrons from photosystem I, making the FLV activity for protection from overreduction superfluous or even detrimental for photosynthetic efficiency. [ABSTRACT FROM AUTHOR]

Although light is the source of energy for photosynthetic organisms, it causes oxidative stress when in excess. Plants and algae prevent reactive oxygen species ( ROS) formation by activation of nonphotochemical quenching ( NPQ), which dissipates excess excitation energy as heat. Although NPQ is found in both algae and plants, these organisms rely on two different proteins for its activation, Light harvesting complex stress-related (LHCSR) and Photosystem II subunit S (PSBS). In the moss Physcomitrella patens, both proteins are present and active., Several P. patens lines depleted in or over-expressing PSBS and/or LHCSR at various levels were generated by exploiting the ability of Physcomitrella to undergo homologous recombination., The analysis of the transgenic lines showed that either protein is sufficient, alone, for NPQ activation independently of the other, supporting the idea that they rely on different activation mechanisms. Modulation of PSBS and/or LHCSR contents was found to be correlated with NPQ amplitude, indicating that plants and algae can directly modulate their ability to dissipate energy simply by altering the accumulation level of one or both of these proteins., The availability of a large range of P. patens genotypes differing in PSBS and LHCSR content allowed comparison of their activation mechanisms and discussion of implications for the evolution of photoprotection during land colonization. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms respond to strong illumination by activating several photoprotection mechanisms. One of them, non-photochemical quenching (NPQ), consists in the thermal dissipation of energy absorbed in excess. In vascular plants NPQ relies on the activity of PSBS, whereas in the green algae Chlamydomonas reinhardtii it requires a different protein, LHCSR. The moss Physcomitrella patens is the only known organism in which both proteins are present and active in triggering NPQ, making this organism particularly interesting for the characterization of this protection mechanism. We analysed the acclimation of Physcomitrella to high light and low temperature, finding that these conditions induce an increase in NPQ correlated to overexpression of both PSBS and LHCSR. Mutants depleted of PSBS and/or LHCSR showed that modulation of their accumulation indeed determines NPQ amplitude. All mutants with impaired NPQ also showed enhanced photosensitivity when exposed to high light or low temperature, indicating that in this moss the fast-responding NPQ mechanism is also involved in long-term acclimation. [ABSTRACT FROM AUTHOR]