A Genetic Screen to Identify New Molecular Players Involved in Photoprotection Qh in Arabidopsis Thaliana

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Article A Genetic Screen to Identify New Molecular Players Involved in Photoprotection qH in Arabidopsis thaliana

Pierrick Bru , Sanchali Nanda and Alizée Malnoë *

Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 901 87 Umeå, Sweden; [email protected] (P.B.); [email protected] (S.N.) * Correspondence: [email protected]

Received: 1 October 2020; Accepted: 11 November 2020; Published: 13 November 2020

Abstract: Photosynthesis is a biological process which converts light energy into chemical energy that is used in the Calvin–Benson cycle to produce organic compounds. An excess of light can induce damage to the photosynthetic machinery. Therefore, plants have evolved photoprotective mechanisms such as non-photochemical quenching (NPQ). To focus molecular insights on slowly relaxing NPQ processes in Arabidopsis thaliana, previously, a qE-deficient line—the PsbS mutant—was mutagenized and a mutant with high and slowly relaxing NPQ was isolated. The mutated gene was named suppressor of quenching 1, or SOQ1, to describe its function. Indeed, when present, SOQ1 negatively regulates or suppresses a form of antenna NPQ that is slow to relax and is photoprotective. We have now termed this component qH and identified the plastid lipocalin, LCNP, as the effector for this energy dissipation mode to occur. Recently, we found that the relaxation of qH1, ROQH1, protein is required to turn off qH. The aim of this study is to identify new molecular players involved in photoprotection qH by a whole genome sequencing approach of chemically mutagenized Arabidopsis thaliana. We conducted an EMS-mutagenesis on the soq1 npq4 double mutant and used chlorophyll fluorescence imaging to screen for suppressors and enhancers of qH. Out of 22,000 mutagenized plants screened, the molecular players cited above were found using a mapping-by-sequencing approach. Here, we describe the phenotypic characterization of the other mutants isolated from this genetic screen and an additional 8000 plants screened. We have classified them in several classes based on their fluorescence parameters, NPQ kinetics, and pigment content. A high-throughput whole genome sequencing approach on 65 mutants will identify the causal mutations thanks to allelic mutations from having reached saturation of the genetic screen. The candidate genes could be involved in the formation or maintenance of quenching sites for qH, in the regulation of qH at the transcriptional level, or be part of the quenching site itself.

Keywords: photoprotection; non-photochemical quenching qH; Arabidopsis thaliana; forward genetics; whole genome sequencing

1. Introduction Photosynthesis is the biological process by which photosynthetic organisms convert sunlight energy into chemical energy. Photosynthesis is the primary process that provides energy for plant growth. ATP and NADPH are the ﬁnal products of photosynthesis that power the Calvin–Benson cycle to produce organic compounds that are used by plant cells for metabolism to support their physiological growth. However, photosynthesis is also a source of damaging reactive oxygen species (ROS) in plants. As a consequence, plants limit their photosynthetic processes to avoid cell damage by ROS. Cell damage would result in a decrease of photosynthetic eﬃciency and thereby the production

Plants 2020, 9, 1565; doi:10.3390/plants9111565 www.mdpi.com/journal/plants Plants 2020, 9, 1565 2 of 14 of organic compounds via the Calvin–Benson cycle will decrease. Furthermore, climate change is exposing plants to more frequent abiotic stresses, such as fluctuating light intensity and drought, which can affect light usage efficiency in plants [1]. Hence, plants have evolved mechanisms to detoxify ROS which involve carotenoids [2,3]. Another solution is to limit the production of ROS by limiting light absorption. When light absorption exceeds photosynthetic capacity, excess light energy is dissipated as heat (also known as non-photochemical quenching NPQ) or as fluorescence [4]. Research on photoprotection is important to understand how its molecular mechanisms function and to find new avenues for plant improvement. Because heat is difficult to measure directly as it dissipates on time and space scales beyond the resolution of available instrumentation, the measurement of chlorophyll (Chl) fluorescence is used to assess NPQ [5]. There is an inverse relationship between Chl fluorescence and NPQ when photochemistry is blocked by a light saturating pulse. The dissipation of excess light energy in the form of heat is measured as a decrease in Chl fluorescence and is termed as NPQ [6]. Fluorescence measurement to assess NPQ is first performed on dark-acclimated samples to assay the minimal fluorescence (Fo) when all the photosystem II (PSII) reaction centers are open and the maximal fluorescence (Fm) when all the reaction centers are closed after flashing a saturating light pulse. Actinic light is then used to induce NPQ, followed by a period of darkness to relax NPQ. Maximal Chl fluorescence is measured during the illumination and dark periods at different time points after a saturating light pulse (Fm’). The PSII quantum yield (Fv/Fm) in the dark can be calculated as (Fm Fo)/Fm. NPQ induction and relaxation is calculated as (Fm Fm’)/Fm’ at different timepoints − − throughout the illumination and dark periods [5]. Several NPQ processes have been identified and reported in the literature. qM, for movement, accounts for the decrease in fluorescence due to chloroplast movements [7]. qT, for state transition, accounts for a fluorescence decrease due to the movement of phosphorylated antenna proteins away from PSII [8,9]. qE, for energy-dependent quenching, results in creation of a quenching site by PsbS in alliance with zeaxanthin which leads to NPQ via heat dissipation. qE relies on the pH gradient across thylakoid membrane and has fast induction and relaxation processes which ranges from seconds to minutes [10–12]. qZ is a zeaxanthin-dependent NPQ process which also leads to heat dissipation and is slow to relax ranging from minutes to tens of minutes [13,14]. Photoinhibition is defined as the light-induced decrease in CO2 fixation and can be due to inactivation and/or destruction of the D1 protein in PSII as well as slowly relaxing NPQ mechanisms [15]. qI is a slow-relaxing process, which accounts for photoinhibitory quenching due to D1 photoinactivation that relaxes in hours or longer [16,17]. However, not all photoinhibition is due to qI and other photoprotective slowly relaxing processes, such as qZ [13,14] and the newly discovered qH, also exist [15,18]. To study slowly relaxing photoprotective NPQ mechanism, Brooks et al. [19] performed a suppressor screen on npq4 gl1 in Arabidopsis thaliana background using ethyl methanesulfonate (EMS) as a chemical mutagen. EMS has an alkylating effect that mainly induces G/C-to-A/T transitions [20,21]. These point mutations have the potential to produce loss of function mutants but also leaky alleles [22]. Chemical mutagenesis is a powerful method to produce new mutants to perform forward genetic analysis. A secondary chemical mutagenesis on a mutant in a particular pathway allows identification of other genes involved in that same pathway [23,24]. After the second mutagenesis, the phenotype can be either enhanced or suppressed compared to the primary phenotype caused by the first mutation [23]. Enhancer mutations would identify redundant gene or mutant gene product that physically interact with the primary mutated gene. Suppressor mutants would identify interacting proteins or alternative pathways activated by the second mutation [24]. Arabidopsis thaliana npq4 mutant lacks the PsbS protein eliminating the occurrence of qE [11,25]. The gl1 (glabrous 1) mutation allows identification of potential contamination from non-mutagenized seeds in the mutant (M) population as gl1 causes lack of trichomes [26]. This suppressor screen led to the discovery of a new mutant impaired in NPQ phenotype. The mutant npq4 soq1 (suppressor of quenching 1) displays higher NPQ than npq4 which slowly relaxes. The SOQ1 protein is a negative regulator of a slowly relaxing NPQ component Plants 2020, 9, 1565 3 of 14 which is independent of PsbS, ∆pH and zeaxanthin formation, STN7-protein phosphorylation and D1 damage/photoinhibition [19]. To address the question of what are the partners of SOQ1 in this photoprotective mechanism, Malnoë et al. [18] performed a second EMS screen on the Arabidopsis thaliana soq1 npq4 gl1 mutant background and searched for mutants that went back to displaying a low NPQ phenotype similar to npq4. In doing so, the protein LCNP (lipocalin in the plastid) was found to be a positive regulator of this quenching mechanism [18]. Following this study, this qI-type quenching has been named qH to differentiate it from quenching due to photodamage as opposed to photoprotection; the letter ‘H’ was chosen for its position in the alphabet before ‘I’, in analogy to protection preceding damage. qH accounts for a slowly relaxing photoprotective quenching in the peripheral antenna of PSII [18]. Although qI, qZ and qH constitute the photoinhibitory processes; individually they are very distinct in their mode of action. While qI is a photoinactivation process due to damage or destruction of D1 in PSII. qZ and qH do not stem from photosystem damage, rather they work in a photoprotective manner [13–16]. In the second round of suppressor screen performed by Malnoë et al. [18], approximately 150 mutants impaired in NPQ and/or Fo,Fm,Fv/Fm were selected. Those mutants display different NPQ phenotype, such as higher or lower NPQ, different Fo,Fm,Fv/Fm, and different pigmentation compared to the parental line soq1 npq4 gl1 mutant. Some of these mutants have been characterized such as lcnp, chlorina1 (cao mutant) [18] and roqh1 (relaxation of quenching 1)[27]. The chlorina1 mutant does not accumulate Chl b and by consequence lacks the PSII peripheral antennae [28]. Moreover, qH is abolished in chlorina1 indicating that qH occurs in the PSII peripheral antennae [18]. The ROQH1 protein is required for relaxation of qH possibly by directly recycling the quenching sites to a light harvesting state [27]. The lcnp, cao, and roqh1 mutations have been identified through a mapping-by-sequencing approach [18,27]. Mapping-by-sequencing is an efficient method to identify causal mutation but is time-consuming due to the necessary backcrosses with the parental line. To accelerate the identification of the causal mutations for the phenotype of the remaining mutants, a direct whole-genome-sequencing approach will be used. Indeed, the low cost of sequencing allows to sequence a large number of mutants. Sequencing a large number of mutants with a similar phenotype can be used to retrieve the mutated gene by finding allelic mutations in the same gene [29–32]. The goal of this study is to categorize the isolated mutants for potential allelism thereby facilitating downstream analysis of the whole genome sequencing data. Here, we present the fluorescence phenotypes of the remaining mutants from the aforementioned suppressor screen and discuss the possible candidate genes causing their phenotype.

2. Results

2.1. Selection of 150 Mutants from the Genetic Screen on soq1 npq4 gl1 A forward genetic screen was performed to identify new molecular players involved in qH: soq1 npq4 gl1 seeds were chemically mutagenized using EMS and sown in 20 pools [18]. Sowing in different pools is important to determine at a later stage if mutants with a similar phenotype may have come from the same mutation event. Indeed, mutants with a similar phenotype coming from the same pool are most likely siblings while mutants from a different pool with a similar phenotype are likely allelic mutants (i.e., mutated in the same gene but with a different mutation). The M1 mutants (1st generation after EMS mutagenesis) were harvested by pools and approximately 30,000 seeds were plated. The resulting seedlings were screened by chlorophyll fluorescence imaging to select photosynthetic and NPQ impaired mutants. The M2 (2nd generation after EMS mutagenesis) selected mutants were grown to propagate the seeds and verify the NPQ phenotype. After this step, 150 mutants were selected. Those mutants display different NPQ phenotypes such as higher or lower NPQ, different Fv/Fm and/or different pigmentation compared to the parental line soq1 npq4 gl1. The mutants with a phenotype that went back to the original NPQ phenotype of the npq4 mutant or that showed constitutive low Fm with no visible pigment defect were back-crossed with the parental line soq1 Plants 2020, 9, 1565 4 of 14 npq4 gl1 in order to identify the causal mutation by mapping-by-sequencing (Figure1A,B). The choice of studied mutants was prioritized on the basis of full suppression (as opposed to intermediate) or highest likelihood to possess constitutive qH (thereby pointing to a major regulator). Out of this mapping-by-sequencing approach, lcnp, chlorina1 (cao mutant), and roqh1 have been identified and characterized [18,27]. To accelerate the identification of the causal mutations in the remaining mutants with an incomplete return to a npq4 phenotype (intermediate lower NPQ) or displaying enhancement of NPQ together with or without a pigmentation defect, a whole-genome-sequencing approach will be used (Figure1C). To facilitate this approach, we have categorized the selected mutants by their NPQ phenotype.

A Genetic screen B Mapping-by-sequencing x Self-cross EMS x M2 soq1 npq4 F1 F2 100 Chr4 80 60 soq1 npq4 40 20 0 gDNA 0 5 10 15 20 Chr2 extraction 100 80 ncy ncy of detection SNP Pool 1 e 60 40 20 Illumina Frequ 0 F2 n>70 sequencing 0 5 10 15 20 25 or Position (Mb) Allelic approach by whole genome sequencing Pool 2 C Self-cross Pool 1 Pool 20 Pool 1 Pool 20 … gDNA Self-cross Pool 1 extraction Pool 2 Pool 2 Pool 1 Illumina Pool 20 M2 M3 n>30 sequencing

Pool 1 Pool 1 M1 M2 Pool 20 Pool 2 soq1 npq4 soq1 npq4 Chr4 Chr2

Figure 1. Identification of new genes involved in qH. (A) soq1 npq4 gl1 seeds were chemically mutagenized and sown in 20 pools. 30,000 seedlings from the M2 generation were screened using chlorophyll fluorescence imaging. Purple or gold colors correspond to mutants impaired in qH. (B) Identification of the mutation by mapping-by-sequencing. The M2 mutants were backcrossed with the parent soq1 npq4 gl1. In the F2 generation, in the case of recessive alleles, 25% will be homozygous mutant (purple or gold), 25% will be homozygous wild-type (green) and 50% will be heterozygous (purple or gold and green stripe colored). F2 homozygous mutants are collected (the larger the number, the narrower the peak; at least n > 70 F2 individuals is advised) to extract gDNA and perform whole genome sequencing. The candidate genes position on the chromosome is revealed where the single-nucleotide polymorphisms (SNPs) frequency equals 100%. (C) The sequencing of potential allelic mutants showing a similar phenotype (represented by the same color) from different pools will facilitate the identification of mutations (here a smaller number of mutant individuals is sufficient e.g., n > 30 M3). The direct whole-genome-sequencing approach will lead to the identification of SNPs (represented by the colored sticks). The red sticks represent the mutations already present in the parental line soq1 npq4 gl1. The blue sticks represent the new mutations. The boxed blue sticks represent the potential allelic mutations and candidate genes.

2.2. Three Classes of Mutants “Lower NPQ”, “Higher NPQ” and “Faster Relaxation” Can Be Distinguished The 150 mutants selected from the screen on soq1 npq4 display different NPQ kinetics and photosynthetic parameters (e.g., Fo,Fm, and Fv/Fm). Among the 150 mutants selected, some mutants were coming from the same pool and displayed the same phenotype. We decided to keep one mutant per pool with the same phenotype to sequence a maximum of 96 mutants. The M3 mutants (3rd generation after EMS mutagenesis) were grown and re-phenotyped by chlorophyll fluorescence imaging for Fo,Fm, Fv/Fm, and NPQ to confirm the phenotype observed in the M2 generation. Three major classes can be Plants 2020, 9, 1565 5 of 14 distinguished from those mutants with one class displaying a lower NPQ level, another one displaying a higher NPQ level and a third class with a NPQ phenotype similar as soq1 npq4 but that relaxes faster; mutants were further classified based on their Fo and/or Fm values (Figure2 and Table S1). Plants 2020, 9, x FOR PEER REVIEW 5 of 14

FigureFigure 2. 2. DistributionDistribution of of“lower “lower NPQ”, NPQ”, “higher “higher NPQ” NPQ” and “faster and “faster relaxation” relaxation” classes classes of mutants. of mutants. Pie chartPie chart representing representing the percentage the percentage of each of each mutant mutant class. class. Mutant Mutant classification classiﬁcation is based is based on onNPQ, NPQ, Fv F/Fvm/F m andand pigmentation pigmentation phenotype. phenotype. Classe Classess marked marked by by an an asterisk asterisk (*) (*) are are further further described described in inthis this study. study.

TheThe different different classes classes are are not not represented represented in in the the same same proportion. proportion. Indeed, Indeed, the the class class with with a “lower a “lower NPQ”NPQ” in in blue blue is is more more represented represented with with approxim approximatelyately 56% 56% of ofthe the total total mutants. mutants. The The “higher “higher NPQ” NPQ” classclass in in orange orange represent represent approximately approximately 32% 32% while while the the “faster “faster relaxation” relaxation” class class in ingreen green represents represents approximatelyapproximately 11% 11% of of the the total total mutants mutants (Figure (Figure 2).2). Within Within these these three three major major classes, classes, subclasses subclasses can can bebe distinguished distinguished with with one one or or more more impaired impaired photosynthetic photosynthetic parameters parameters (e.g., (e.g., NPQ, NPQ, Fo F, oF,Fm, mF,Fv/Fvm/).Fm ). WithinWithin these these subclasses, subclasses, mutants mutants with with a apigmentati pigmentationon deficiency deficiency have have also also been been identified identified (Figure (Figure 2 2 andand Table Table S1). S1).

2.3.2.3. The The Normal Normal Green, Green, Low Low NPQ NPQ and and Low Low Fv F/Fv/mF mduedue to toHigh High Fo FMutanto Mutant Class Class TheThe mutants mutants No.36 and No.39No.39 fromfrom thethe pools pools 6 6 and and 14 14 respectively respectively display display about about 33% 33% lower lower NPQ NPQinduction induction after 10after min 10 of min high of light high compared light compared to soq1 to npq4 soq1(Figure npq4 3(FigureA). The 3A). photosynthetic The photosynthetic parameter parameterFv/Fm of 0.49 Fv/F andm of 0.470.49 isand due 0.47 to ais highdue to Fo aof high 257 Fo 13of and257 ± 264 13 and8, respectively264 ± 8, respectively compared compared to a Fv/F tom of ± ± a0.79 Fv/F andm of F 0.79o of 108and Fo6 of for 108soq1 ± npq46 for (Figuresoq1 npq43B, (Figure Supplementary 3B, Supplementary Table S2). The Table visual S2). leafThe pigmentation visual leaf ± pigmentationis normal green is normal to slightly green pale to greenslightly compared pale green to soq1compared npq4 (Figure to soq13 D).npq4 To (Figure further 3D). characterize To further the characterizepigmentation, the Chlpigmentation, content and Chla/b contentratio have and beena/b ratio measured. have been The measured. mutants The No.36 mutants and No.39 No.36 display and No.39a slightly display lower a slightly Chl a/b lowerof 2.7 Chl compared a/b of 2.7 to compared 3.0 for soq1 to npq43.0 forbut soq1 overall npq4 but have overall a similar have Chla similar content Chlto the content control to the (Figure control3C). (Figure The mutants 3C). The No.36 mutants and No.36 No.39 and display No.39 a similardisplay Fa vsimilar/Fm,Fo F,v and/Fm, NPQFo, and and NPQcome and from come diﬀerent from pools. different Therefore, pools. those Therefore, mutants those could mutants be allelic could for the be NPQ allelic phenotype. for the TheNPQ Chl phenotype.phenotype The is also Chl similar phenotype and isis likelyalso similar linked and to the is likely NPQ phenotype.linked to the Five NPQ other phenotype. mutants Five were other found mutantswith a similar were found phenotype with a (Supplementarysimilar phenotype Table (Supplementary S1) and could Table be other S1) and mutant could alleles be other aﬀ ectingmutant the allelessame geneaffecting as No.36 the same and gene No.39. as No.36 and No.39.

Plants 2020, 9, 1565 6 of 14 Plants 2020, 9, x FOR PEER REVIEW 6 of 14

AB 2.0 600 soq1 npq4 0.8 No.39 1.5 soq1 npq4 400 No.36 0.6 * * 1.0 **

NPQ 0.4 No.39 200 0.5 No.36 Fluorescence 0.2

0.0 0 0.0 0 5 10 15 20 Fo Fm Fv/Fm Time (min)

C ) D soq1 npq4 No.39 No.36 1.0 soq1 npq4

0.5 Chlorophyll content (relative to to (relative 0.0 3.0 ± 2.7 ±**2.7 ± Chl a/b 0.12 0.04 0.02 4 q 9 6 p .3 n o 1 N No.3 1 cm q o s

Figure 3. Normal green, low NPQ and low F /F due to high F , potential allelic mutants. (A) NPQ Figure 3. Normal green, low NPQ and lowv Fv/Fm m due to high oFo, potential allelic mutants. (A) NPQ kineticskinetics of dark-acclimatedof dark-acclimated (20 (20 min) min)soq1 soq1 npq4, npq4, No.36 No.36 and No.39and No.39three-week-old three-week-old plants plants grown grown at 120 atµmol 120 2 1 2 1 photonsµmol photons m− s− m.−2 Induction s−1. Induction of NPQ of NPQ at 1300at 1300µmol µmol photons photons m m− −2 ss−−1 (white(white bar), bar), and and relaxation relaxation in in the dark (black bar). Data represent means SD (n = 3 individuals and 2 measures per the dark (black bar). Data represent means +/− SD (±n = 3 individuals and 2 measures per individuals). individuals).(B) Photosynthetic (B) Photosynthetic parameters Fo, parameters Fm and Fv/F Fm oof,F plantsm and used Fv/ Finm A.of No.36 plants and used No.39 in are A. statisticallyNo.36 and

No.39identicalare statistically but different identical from soq1 but npq4 diﬀ erentfor Fo; from p < 0.0001.soq1 npq4 No.36for, No.39 Fo; p and< 0.0001. soq1 npq4No.36 are, statisticallyNo.39 and

soq1identical npq4 are for statistically Fm. Fv/Fm between identical No.36 for,F No.39m.Fv is/F mstatisticallybetween No.36identical, No.39 but isdifferent statistically from identicalsoq1 npq4 but p < different from soq1 npq4 p < 0.0001. Data represent means SD (n = 3 individuals and 2 measures per 0,0001. Data represent means +/− SD (n = 3 individuals and± 2 measures per individuals). (C) Relative individuals).Chl content ( Cto) soq1 Relative npq4 Chl and content Chl a/b toratiosoq1. Chl npq4 contentand Chl wasa/ bnormalizedratio. Chl contentby leaf fresh was normalized weight. No.36 by, leafNo.39 fresh and weight. soq1 No.36npq4 are, No.39 statisticallyand soq1 identical npq4 are for statistically Chl content. identical No.36 for, No.39 Chl content.are statisticallyNo.36, No.39 identicalare statisticallyfor Chl a/b identical but statistically for Chl a /bdifferentbut statistically from soq1 di ffnpq4erent p from< 0.02soq1 (No.36) npq4 p <= 0.020.01 ((No.39).No.36) p Data= 0.01 represent (No.39). Data represent means SD (n = 3 individuals). (B,C) ANOVA Tukey’s multiple comparison was used means +/− SD (n= 3 ±individuals). (B,C) ANOVA Tukey’s multiple comparison was used with a 95% withconfidence a 95% confidence interval intervalfor statistical for statistical analysis analysis (with GraphPad (with GraphPad Prism Prismsoftware). software). (D) Image (D) Image of one of onerepresentative representative detached detached leaf leaf from from three- three-week-oldweek-old plants plants amon amongg 3 individuals. 3 individuals. * p *value.p value.

2.4. The Pale Green, Low NPQ and Lower Fv/Fm Mutant Class 2.4. The Pale Green, Low NPQ and Lower Fv/Fm Mutant Class The mutants No.37 and No.245 from pools 6 and 14 respectively display about 50% lower NPQ The mutants No.37 and No.245 from pools 6 and 14 respectively display about 50% lower NPQ induction after 10 min of high light and a visible pigmentation defect compared to the control soq1 induction after 10 min of high light and a visible pigmentation defect compared to the control soq1 npq4 (Figure4A,D). To further characterize the pigmentation defect, Chl content and a/b ratio have npq4 (Figure 4A,D). To further characterize the pigmentation defect, Chl content and a/b ratio have beenbeen measured. measured. The The mutants mutants display display an an abnormal abnormal Chl Chl a/b a/b ratio ratio of 7.2of 7.2 and and 6.9 6.9 respectively respectively compared compared to 3.1to for 3.1soq1 for soq1 npq4. npq4.In addition, In addition, No.37 No.37 and and No.245 No.245 display display a decreasea decrease in in Chl Chl content content of of 70%70% andand 67%67% respectivelyrespectively compared compared to tosoq1 soq1 npq4 npq4(Figure (Figure4 4C,D)C,D)close close toto thethe 75% decrease decrease in in chlorophyll chlorophyll content content of ofthe the pale green par excellenceexcellence mutantmutant chlorina1-1chlorina1-1 (compared(compared toto wildwild type)type) [[33].33]. TheThe photosyntheticphotosynthetic parameters, F and F values are also affected with a statistically lower F and a statistically higher parameters,o Fo and mFm values are also affected with a statistically lower Fmm and a statistically higher Fo Focomparedcompared to to soq1soq1 npq4 npq4.. This This lower lower F m andand higher F o resultresult in lower Fv/F/Fm valuesvalues of of 0.55 and 0.59, respectively,respectively, compared compared to to 0.79 0.79 for forsoq1 soq1 npq4 npq4(Figure (Figure4B, 4B, Supplementary Supplementary Table Table S2). S2). The The mutants mutants No.37 No.37 andand No.245 No.245 display display a similara similar phenotype phenotype for for NPQ, NPQ, F oF,Fo, mFm,, and and F Fvv//FFmm,, andand comecome from didifferentfferent pools. Therefore,Therefore, those those mutants mutants could could be be allelic allelic for for the the NPQ NPQ phenotype. phenotype. TheThe ChlChl phenotypephenotype isis alsoalso veryvery similarsimilar and and is is likely likely linked linked to to the the NPQ NPQ phenotype. phenotype. No No other other mutants mutants were were foundfound withwith anan identicalidentical phenotype,phenotype, but but 14 mutants14 mutants were were found found with with a similar a similar phenotype phenotype (Supplementary (Supplementary Table S1,Table pale S1, green, pale lowgreen, NPQ, low low NPQ, Fv/F mlow) and Fv/F couldm) and be could other be mutant other mutant alleles aallelesffecting affecting the same the gene same or gene pathway or pathway as No.37 as andNo.37 No.245. and No.245.

Plants 2020, 9, 1565 7 of 14 Plants 2020, 9, x FOR PEER REVIEW 7 of 14

AB 2.5 600 soq1 npq4 0.8 No.37 2.0 soq1 npq4 ** ** 400 No.245 0.6 1.5 Fv/Fm

NPQ 0.4 1.0 No.37 200 ** Fluorescence 0.5 0.2 No.245 0.0 0 0.0 0 5 10 15 20 Fo Fm Fv/Fm Time (min)

C ) D soq1 npq4 No.37 No.245 1.0 soq1 npq4

0.5 ** Chlorophyll content (relative to 0.0 3.1 ± 7.2 ± * 6.9 ±* Chl a/b 0.02 0.17 0.12 4 q 45 p 2 n . 1 No.37 1 cm q No o s

FigureFigure 4. 4.PalePale green, green, low lowNPQ NPQ and lower and lower Fv/Fm,F potentialv/Fm, potential allelic mutants. allelic mutants. (A) NPQ (kineticsA) NPQ of kinetics dark- of acclimateddark-acclimated (20 min) (20 soq1 min) npq4,soq1 No.37 npq4, No.37and No.245 and No.245 three-week-oldthree-week-old plants plants grown grown at 120 at µmol 120 µ molphotons photons 2 1 2 1 m−m2 s−−1.s Induction− . Induction of NPQ of NPQ at 1300 at 1300 µmolµmol photons photons m−2m s−−1 (whites− (white bar), bar),and relaxation and relaxation in the in dark the dark (black (black bar).bar). Data Data represent represent meansmeans +/SD− (SDn = (3n individuals= 3 individuals and 2 measuresand 2 measures per individuals). per individuals). (B) Photosynthetic (B) ± Photosyntheticparameters F oparameters,Fm and FFvo/,F mFmof and plants Fv/Fm used of plants in A. usedNo.37 inand A. No.37No.245 andare No.245 statistically are statistically identical but identicaldiﬀerent but from differentsoq1 npq4 fromfor soq1 Fo ;npq4p < 0.0001 for Fo;. pNo.37 < 0.0001.and No.37No.245 andare No.245 statistically are statistically identical but identical diﬀerent but from

differentsoq1 npq4 fromfor soq1Fm; pnpq4< 0.0001 for Fm (;No.37 p < 0.0001) and (PNo.37= 0.0002) and (No.245). P= 0.0002F v(/No.245).Fm between Fv/FmNo.37 between, No.245 No.37and, No.245soq1 npq4 andare soq1 diff npq4erent arep < different0.0001. Data p < 0.0001. represent Data means representSD means (n = 3 +/- individuals SD (n = 3 andindividuals 2 measures and per 2 measures individuals). ± per(C individuals).) Relative Chl (C content) Relative to soq1 Chl npq4contentand to Chl soq1a/ bnpq4ratio. and Chl Chl content a/b ratio was. Chl normalized content was by leaf normalized fresh weight. byNo.37 leaf freshand weight.No.245 areNo.37 statistically and No.245 identical are statistically but diff erentidentical from butsoq1 different npq4 for from Chl soq1 content; npq4 forp < Chl0.0001 . content;No.37, pNo.245 < 0.0001and. No.37soq1, npq4 No.245are and statistically soq1 npq4 di ffareerent statistically from each different other; p from< 0.0001 each ( soq1other; npq4, p < 0.0001 No.37 and (soq1No.245) npq4, p No.37= 0.02 and(No.37 No.245)and No.245 p = 0.02).Data (No.37 represent and No.245). means DataSD represent (n = 3 individuals). means +/− (SDB,C ()n ANOVA = 3 ± individuals).Tukey’s multiple (B,C) ANOVA comparison Tukey’s was multiple used with comparison a 95% confidence was used interval with a for95% statistical confidence analysis interval (with forGraphPad statistical Prismanalysis software). (with GraphPad (D) Image Prism of one software). representative (D) Image detached of one representative leaf from three-week-old detached leaf plants fromamong three-week-old 3 individuals. plants * p value.among 3 individuals. * p value.

2.5. The Pale Green, High NPQ and Normal Fv/Fm Mutant Class 2.5. The Pale Green, High NPQ and Normal Fv/Fm Mutant Class TheThe mutants mutants No.73 No.73 and and No.251 No.251 from from pools pools 12 12and and 9 respectively 9 respectively display display about about 40% 40% enhanced enhanced NPQNPQ induction induction after after 10 10min min of ofhigh high light light and and a visible a visible pigmentation pigmentation defect defect compared compared to the to thecontrol control soq1soq1 npq4 npq4 (Figure(Figure 5A,D).5A,D). To To further characterizecharacterize thethe pigmentationpigmentation defect, defect, Chl Chl content content and anda /ab/bratio ratio have havebeen been measured. measured. Mutant Mutant No.251 No.251 fully fully developed developed leaves leaves display display a wild-type a wild-type Chl Chla/b ratioa/b ratio of 3.1 of but3.1 the butChl the content Chl content is decreased is decreased by 45% by (Figure 45% (Figure5C,D). No.2515C,D). No.251 younger younger leaves showleaves a show more drastica more paledrastic green palephenotype green phenotype that tends that to disappear tends to disappear with leaf age with (Supplementary leaf age (Supplementary Figure S1). Figure The mutant S1). The No.73 mutant displays No.73a slightly displays lower a slightly Chl a/b ratiolower of Chl 2.8 anda/b ratio a decrease of 2.8 ofand 60% a decrease in Chl content of 60% so in these Chl mutantscontent areso these less pale mutantsgreen thanare lesschlorina1 pale green. The than photosynthetic chlorina1. The parameters photosynthetic Fo and parameters Fm are statistically Fo and Fm lower are statistically compared to lowersoq1 compared npq4 but result to soq1 in npq4 a wild-type but result value in a of wild-type Fv/Fm of value 0.8 (Figure of Fv/F5B,m of Table 0.8 (Figure S2). The 5B, mutants Table S2). No.73 The and mutantsNo.251 No.73 display and a similar No.251 phenotype display a for similar NPQ, phenotype Fo,Fm,Fv/F form, andNPQ, come Fo, fromFm, F div/Fffmerent, and pools. come Therefore, from differentthose mutants pools. Therefore, could be allelic those formutants the NPQ could phenotype. be allelic for The the Chl NPQ phenotype phenotype. is very The similar Chl phenotype and is likely is linkedvery similar to the NPQand phenotype.is likely linked The Chlto the phenotype NPQ phenotype. slight diff erencesThe Chl between phenotype the mutantsslight differences could be due betweento a weaker the mutants and a stronger could be allele. due to Five a weaker other mutantsand a stronger were found allele. withFive aother similar mutants phenotype were found (Table S1) withand a could similar be phenotype other mutant (Table alleles S1) a ffandecting could the be same other gene mutant as No.73 alleles and affecting No.251. the same gene as No.73 and No.251.

Plants 2020, 9, 1565 8 of 14 Plants 2020, 9, x FOR PEER REVIEW 8 of 14

AB 600 4.0 soq1 npq4 ** 0.8 No.251 No.251 3.0 400 No.73 0.6 Fv/Fm No.73 2.0 0.4 NPQ soq1 npq4 200 1.0 Fluorescence ** 0.2

0.0 0 0.0 0 5 10 15 20 Fo Fm Fv/Fm Time (min)

C ) D soq1 npq4 No.251 No.73 1.0

soq1 npq4 * 0.5 * Chlorophyll content (relative to to (relative 0.0 * 3.1 ± 3.1 ± 2.8 ± Chl a/b 0.02 0.05 0.05 4 q p n .251 1 cm 1 o No.73 q N o s

Figure 5. Pale green, high NPQ and normal F /F , potential allelic mutants. (A) NPQ kinetics Figure 5. Pale green, high NPQ and normal Fv/Fm,v potentialm allelic mutants. (A) NPQ kinetics of dark- ofacclimated dark-acclimated (20 min) (20 soq1 min) npq4,soq1 No.251 npq4, and No.251 No.73and three-week-oldNo.73 three-week-old plants grown plants at grown120 µmol at 120photonsµmol 2 1 2 1 photonsm−2 s−1. Induction m− s− . Inductionof NPQ at of1300 NPQ µmol at 1300photonsµmol m photons−2 s−1 (white m− bar),s− and(white relaxation bar), and in relaxationthe dark (black in the dark (black bar). Data represent means SD (n = 3 individuals and 2 measures per individuals). bar). Data represent means +/− SD (n ±= 3 individuals and 2 measures per individuals). (B) No.251 No.73 (BPhotosynthetic) Photosynthetic parameters parameters Fo, FFom,F andm and Fv/F Fvm/ Fofm plantsof plants used used in inA. A.No.251 andand No.73 areare statistically statistically

identicalidentical butbut didifferentﬀerentfrom fromsoq1 soq1 npq4npq4for for FFoo;; p = 0.0040.004 ( (No.251No.251)) and and pp == 0.0410.041 (No.73 (No.73).). No.251No.251 andand No.73No.73

areare statisticallystatistically identicalidentical but didifferentfferent from soq1 npq4 for F m;; pp == 0.0040.004 (No.73 (No.73) )and and pp == 0.0110.011 (No.251 (No.251). ). Fv/Fm between No.251, No.73 and soq1 npq4 is statistically identical. Data represent means SD Fv/Fm between No.251, No.73 and soq1 npq4 is statistically identical. Data represent means +/− SD (±n = (3n individuals= 3 individuals and 2 and measures 2 measures per individuals). per individuals). (C) Relative (C) Relative Chl content Chl content to soq1 tonpq4soq1 and npq4 Chland a/b Chlratioa./ b ratio.Chl content Chl content was normalized was normalized by leaf byfresh leaf weight. fresh weight.No.251, No.73No.251 and, No.73 soq1 npq4and soq1are statistically npq4 are statistically different difromfferent each from other each for otherChl content; for Chl p content;< 0.0003 (No.251p < 0.0003 and(No.251 soq1 npq4and), psoq1 < 0.0001 npq4 (),No.73p < 0.0001and soq1 (No.73 npq4),and p soq1= 0.03 npq4) (No.251, p = 0.03and No.73).(No.251 Chl and a No.73/b ratio). soq1 Chl anpq4/b ratio andsoq1 No.251 npq4 isand statisticallyNo.251 isidentical statistically but No.73 identical is butdifferentNo.73 fromis di ffsoq1erent npq4 from andsoq1 No.251; npq4 pand = 0.0006No.251; (soq1 p npq4)= 0.0006 and (soq1p = 0.0017 npq4) (No.251). and p = Data0.0017 represent (No.251 ). Data represent means SD (n = 3 individuals). (B,C) ANOVA Tukey’s multiple comparison was used means +/− SD (n= 3 individuals).± (B,C) ANOVA Tukey’s multiple comparison was used with a 95% withconfidence a 95% confidenceinterval for interval statistical for statisticalanalysis analysis(with GraphPad (with GraphPad Prism software). Prism software). (D) Image (D) Imageof one of onerepresentative representative detached detached leaf leaffrom from three- three-week-oldweek-old plants plants amon amongg 3 individuals. 3 individuals. * p value. * p value. 3. Discussion 3. Discussion Three classes of mutants have been presented displaying impairment of some or all photosynthetic Three classes of mutants have been presented displaying impairment of some or all parameters studied (Fo,Fm,Fv/Fm and NPQ) combined or not with a different pigment content than photosynthetic parameters studied (Fo, Fm, Fv/Fm and NPQ) combined or not with a different pigment control.content than These control. three setsThese of three mutants sets areof mutants potentially are allelicpotentially (i.e., allelic affecting (i.e., three affecting different three genes) different and havinggenes) and characterized having characterized them will assist them thewill identification assist the identification of the causative of the mutations.causative mutations. In the following In the section,following we section, will discuss we will the discuss putative the genes putative that couldgenes bethat mutated could be and mutated causing and the causing observed the phenotypes. observed phenotypes. 3.1. Less qH Possibly Due To a Deficiency in a Factor Required for PSII Activity

3.1. LessHigh qH F oPossiblycan be Due due To to a either Deficiency PSII corein a Factor inactivation Required or for PSII PSII antenna Activity detachment [34]. If the PSII core is damaged, or less accumulated, or if the PSII antennae are detached from the core, then the High Fo can be due to either PSII core inactivation or PSII antenna detachment [34]. If the PSII low intensity detecting light that measures F cannot be as eﬃciently used for photochemistry and by core is damaged, or less accumulated, or if theo PSII antennae are detached from the core, then the low consequence the light energy re-emitted as Chl ﬂuorescence is higher. Here, No.36 and No.39 display intensity detecting light that measures Fo cannot be as efficiently used for photochemistry and by aconsequence high Fo, low the Fv light/Fm, energy and low re-emitted NPQ phenotype as Chl fluore comparedscence is to higher.soq1 npq4 Here,(Figure No.363 andA,B). No.39 Mutation display in genes encoding factors such as LPA1 [35], PSB33 [36], PAM68 [37], or HCF136 [38] involved in PSII a high Fo, low Fv/Fm, and low NPQ phenotype compared to soq1 npq4 (Figure 3A,B). Mutation in genes encoding factors such as LPA1 [35], PSB33 [36], PAM68 [37], or HCF136 [38] involved in PSII

Plants 2020, 9, 1565 9 of 14

biogenesis, assembly, or stability could be responsible for the high Fo, low Fv/Fm phenotype. However, it is not evident why mutation in these genes would cause less qH. Another candidate gene whose mutation could explain both a lower PSII activity and less qH is LTO1 [39]. Indeed, LTO1 is a disulﬁde bond–forming enzyme in the thylakoid lumen and could oxidize LCNP (which has six conserved cysteines [40]) thereby regulating LCNP function in qH. No.36 and No.39 also display a lower Chl a/b compared to soq1 npq4 (Figure3C,D). A lower Chl a/b could be due to an overaccumulation of Chl b compared to Chl a. This phenotype could be the result of less Chl b degradation typical of the nol and/or nyc1 mutant although they show a wild-type Fv/Fm [41,42].

3.2. Less qH Possibly Due To a Decrease in Quenching Sites

The mutants No.37 and No.245 display a low NPQ with a pale green phenotype and lower Fv/Fm due to a higher Fo and lower Fm compared to soq1 npq4 (Figure4). The pigmentation defect has been assayed by measuring total Chl content and a/b ratio. Chl a is more abundant in the PSII core, while Chl b is more abundant in the antenna therefore a higher Chl a/b ratio can provide an indication of a smaller PSII antenna size [43]. Here, the Chl a/b ratio is 7 in the mutants compared to 3 in the control and the Chl content is decreased by approximately 70%. The lower Chl b content could be due to alteration in Chl metabolism, i.e., less Chl b synthesis or more degradation, lack of Chl insertion, a mutation at a Chl binding site, or fewer antennae due to issues with gene expression or protein biogenesis. This specific phenotype is reminiscent of a cao leaky mutant, chlorina1-2 [44], or a gun4 or gun5 mutation (genome uncoupled 4 and 5). These genes encode for a tetrapyrrole binding protein and a magnesium chelatase respectively which are involved in the chlorophyll biosynthetic process [45–47]. LHCs use cpSRP (chloroplast signal recognition particle) pathway for their targeting to thylakoids. chaos and ffc mutant deficient in cpSRP43 and cpSRP54 respectively display a decrease of Chl content but with a normal Chl a/b ratio [48]. In addition, chaos and ffc display a wild-type Fv/Fm of 0.8 [48]. It is thus likely that No.37 and No.245 are not mutated in the cpSRP pathway as they have a high Chl a/b. The mutations in No.37 and No.245 possibly result in a decreased accumulation of antennae required for qH, thereby explaining the lower NPQ phenotype.

3.3. Enhancement of qH Possibly Due To an Increase in Quenching Sites The mutants No.251 and No.73 display a higher NPQ with a pale green phenotype and a lower Fo and Fm that result in wild-type Fv/Fm of 0.8 (Figure5). Lower F o and Fm values can be due to constitutive quenching in the antenna as was observed for the roqh1 alleles [27] or could be due to less antenna accumulation as was observed for chlorina1 [49]. However, in soq1 npq4 roqh1 and soq1 npq4 chlorina1 mutants, NPQ is lower than soq1 npq4. Here, the mutants No.251 and No.73 display an increased NPQ with a Chl content decreased by approximately 45% and 60%, respectively. This phenotype could be due to a mutation in a gene coding for a Lhcb protein (or a factor involved in LHCB expression) leading to an increase in qH quenching sites as a result of a possible compensatory mechanism between the Lhcbs. Indeed, previous reports support that absence of a specific Lhcb protein leads to a compensatory effect causing an increase in other types of Lhcb and can lead to an abnormal Chl content and a/b ratio. For example, when all isoforms of LHCB1 are knocked down an increase in Lhcb2 and Lhcb3 proteins is observed [50]. The Chl content of a lhcb1 mutant is decreased by 30% and the Chl a/b ratio is equal to 4, compared to 3.2 for wild-type [50]. When LHCB2 is knocked down, Lhcb3 and Lhcb5 protein accumulation is increased. The Chl content and Chl a/b ratio of a lhcb2 mutant is similar to wild-type [50]. Recently, it was reported that when both LHCB1 and LHCB2 are knocked-down, Lhcb5 is not upregulated [51]. Finally, when LHCB3 is knocked-out, an increase of the proteins Lhcb1 and Lhcb2 is observed. No significant differences in the pigment composition of a lhcb3 mutant compared to wild-type is observed [52]. However, the situation is slightly different here: the mutants No.251 and No.73 have similar Chl a/b ratio as control (as in lhcb2 or lhcb3 mutants) but overall decreased Chl content (as in lhcb1 mutant). lhcb 4, 5, or 6 mutations are resulting in a slightly lower Chl a/b, Chl content and Fv/Fm [53–55]. It is thus likely that No.251 and No.73 are not mutated Plants 2020, 9, 1565 10 of 14 in a minor Lhcb (Lhcb4-6). Another explanation for this phenotype could be that a gene involved in chloroplast ultrastructure formation is mutated, leading to a change in the antennae organization which would promote qH. Genetic screens are performed to identify novel genes involved in a pathway or understand cross-talks between pathways. Previously, map-based cloning has been used in Arabidopsis to identify causal mutations such as in the hcef1 mutant affected in the chloroplast fructose-1,6-bisphosphatase [56]. Brooks et al. [19] used this approach on EMS mutagenized qE-deficient mutant line npq4 in a quest to identify other proteins involved in NPQ qI. This study led to the discovery of SOQ1, a repressor of a slowly relaxing NPQ mechanism which is now termed qH. Ensued the identification of the molecular partners of SOQ1 by Malnoë et al. [18] and Amstutz et al. [27] who performed a suppressor screen on soq1 npq4 line and identified by mapping-by-sequencing LCNP and ROQH1 to be involved in qH. Other examples using this approach, also called bulked segregant analysis by whole-genome re-sequencing, are the identification of mutations restoring the photorespiratory defect of er-ant1 [57] or anthocyanin accumulation of tt19 [58]. A direct whole genome sequencing approach has recently proven successful to identify causal mutations of allelic M3 lines by comparison to the parental line in mutants with meiotic defects [30–32]. According to Jander et al. [59], 50,000 M1 lines need to be tested to have 95% chance to find mutation in any G:C in the genome. Malnoë et al. [18] screened 22,000 M2 lines and a further 8000 were screened here ensuring saturation or at least sub-saturation of the screen as two mutant alleles of LCNP, CAO and ROQH1 have been identified [18,24,27]. Indeed, the mutants presented here have two to seven potential alleles meaning that some saturation has been reached (Supplementary Table S1). Future investigation will determine whether LCNP redox status is affected in mutants No.36 and No.39, which antenna proteins may be lacking from the mutants No.37 and No.245 or overaccumulating in the mutants No.251 and No.73 and the causative mutations for their phenotype will be identified using a direct whole genome sequencing approach.

4. Materials and Methods

4.1. Plant Material and Growth Conditions The soq1 npq4 gl1 Arabidopsis thaliana mutant [19] is of the Col-0 ecotype and is mostly referred to as soq1 npq4 in the main text and ﬁgures. The EMS mutants studied here were derived from mutagenesis of soq1 npq4 gl1 seeds [18]. Seeds were surface sterilized using 70% ethanol and sown on MS plates (Murashige and Skoog Basal Salt Mixture, Duchefa Biochemie, with pH adjusted to 5.7 with KOH) and placed for 1 day in the dark at 4 ◦C. Plates are then transferred into a growth cabinet room with 12 h 2 1 light (Philips F17T8/TL741/ALTO 17W) at 150 µmol photons m− s− light intensity and 12 h dark at constant temperature 22 ◦C. Seedlings were then transferred into soil (1:3 mixture of Agra- vermiculite + “yrkeskvalité K-JORD/krukjord” provided by RHP and Hasselfors garden respectively) and placed into 2 1 a short-day growth room 8 h light with 150 µmol photons m− s− light (Philips F17T8/TL841/ALTO 17W) at 22 ◦C and 16 h dark at 18 ◦C. To promote ﬂowering and collect seeds, plants were placed in a 2 1 long-day growth room with 16 h light at 150 µmol photons m− s− light (Philips F17T8/TL841/ALTO 17W) at 22 ◦C and 8 h dark at 18 ◦C.

4.2. Chlorophyll Fluorescence Measurement Detached leaf from different mutant individuals were placed on a plate for 20 min in the dark to relax NPQ. Fluorescence was acquired with the SpeedZen fluorescence imaging setup from JbeamBio [60]. The following script was used to measure Fo,Fm and Fv/Fm and NPQ: 30ms/E0!30µsD20msE13!250msE0!30µsD20msE11!/30sZ10(60sZ)30msE0!15sD20msE13!250msE0!30 µsD20sD20msE13!250msE0!30µsD10(60sD20msE13!250msE0!30µsD). The command E turns on the actinic light at the intensity called by the number, e.g., “E0!” turns of the light for the given time stated after “!”. The measuring sequence involves detection light (D) at level 100. Z calls the repeat written between “/”. Briefly, this script results in the following: the sequence starts in the dark with a first Plants 2020, 9, 1565 11 of 14

2 1 ﬂuorescence measurement (Fo) then follows a saturating pulse at 2600 µmol photons m− s− (E13) to measure Fm.Fv/Fm is calculated as (Fm Fo)/Fm. Then, NPQ is induced for 10 min at 1300 µmol 2 1 − photons m− s− (E11) (red actinic light) and relaxed for 10 min in the dark. Maximum ﬂuorescence levels after dark acclimation (Fm) and throughout measurement (Fm’) were recorded after applying a saturating pulse of light to calculate NPQ as (Fm/Fm0)/Fm0.

4.3. Chlorophyll Extraction Leaves were detached, weighed, and the area was measured. Leaf material was then ﬂash-frozen in liquid nitrogen and ground. Chl was extracted twice by adding 100 µL of 100% acetone, vortexing and centrifuging to remove cell debris. To measure, the Chl content 100 µL of the extract was diluted in 700 µL of 80% cold acetone. The optical density was measured at 647, 664, and 750 nm. Total Chl, Chl a and b contents were calculated using the Porra method [61].

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/11/1565/s1. Figure S1: Image of No.251 mutant whole plant. Table S1: Mutants classification per phenotype. Table S2: Photosynthesis parameters Fo, Fm and Fv/Fm. Author Contributions: Conceptualization, P.B. and A.M.; methodology, P.B.; validation, P.B., S.N. and A.M.; formal analysis, P.B.; investigation, P.B.; resources, A.M.; data curation, P.B. and A.M.; writing—original draft preparation, P.B. and S.N.; writing—review and editing, P.B. and A.M.; visualization, P.B.; supervision, A.M.; project administration, A.M.; funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Division of Chemical Sciences, Geosciences and Biosciences, the Office of Basic Energy Sciences and the Office of Science, US Department of Energy (Field Work Proposal 449B; A.M. while at UC Berkeley) and by a starting grant from the Swedish Research Council Vetenskapsrådet (2018-04150). The APC was funded by a starting grant to A.M. from the Faculty of Sciences and Technology, Umeå University. A.M. was supported by European Commission Marie Skłodowska-Curie Actions Individual Fellowship Reintegration Panel (845687). Acknowledgments: We are grateful to Kris Niyogi, UC Berkeley for his support, guidance and gift of the material generated in his group by A.M. during her postdoctoral training. We warmly thank the Bioresource and Technology (BRT) VT19 students: Julia Avander, Cornelia Byström, Johanna Nilsson, Joacim Nyman, Björn Sundqvist, Martin Sundqvist, Johanna Wuotila from the Dept. of Plant Physiology, Umeå University for their help in further screening the mutant pools and selecting new mutants. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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