Limiting etioplast gene expression induces apical hook twisting during skotomorphogenesis of Arabidopsis seedlings.

When covered by a layer of soil, seedling development follows a dark-specific program (skotomorphogenesis). In the dark, seedlings consist of small, non-green cotyledons, a long hypocotyl, and an apical hook to protect meristematic cells. We recently highlighted the role played by mitochondria in the high energy-consuming reprogramming of Arabidopsis skotomorphogenesis. Here, the role played by plastids, another energy-supplying organelle, in skotomorphogenesis is investigated. This study was conducted in dark conditions to exclude light signals so as to better focus on those produced by plastids. It was found that limitation of plastid gene expression (PGE) induced an exaggerated apical hook bending. Inhibition of PGE was obtained at the levels of transcription and translation using the antibiotics rifampicin (RIF) and spectinomycin, respectively, as well as plastid RPOTp RNA polymerase mutants. RIF-treated seedlings also showed expression induction of marker nuclear genes for mitochondrial stress, perturbation of mitochondrial metabolism, increased ROS levels, and an augmented capacity of oxygen consumption by mitochondrial alternative oxidases (AOXs). AOXs act to prevent overreduction of the mitochondrial electron transport chain. Previously, we reported that AOX1A, the main AOX isoform, is a key component in the developmental response to mitochondrial respiration deficiency. In this work, we suggest the involvement of AOX1A in the response to PGE dysfunction and propose the importance of signaling between plastids and mitochondria. Finally, it was found that seedling architecture reprogramming in response to RIF was independent of canonical organelle retrograde pathways and the ethylene signaling pathway.

Reducing PHYTOENE SYNTHASE activity fine-tunes the abundance of a cis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis.

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4, psy-90, psy-130, and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic post-transcriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or ß-carotene accumulation in ccr2, yet they reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development.

HSP90s are required for hypocotyl elongation during skotomorphogenesis and thermomorphogenesis via the COP1-ELF3-PIF4 pathway in Arabidopsis.

Light and temperature are two key environmental signals that share several molecular components that, in turn, regulate plant growth. Darkness and high ambient temperatures promote skoto- and thermomorphogenesis, including stem elongation. Heat shock proteins 90 (HSP90s) facilitate the adaptation of organisms to various adverse environmental stimuli. Here, we showed that HSP90s are required for hypocotyl elongation during both skoto- and thermomorphogenesis. When HSP90s activities are impaired by the knockdown of HSP90s expression or the application of HSP90 inhibitors, the expression levels and protein abundance of PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) markedly decreased. EARLY FLOWERING 3 (ELF3) deficiency was resistant to the inhibition of HSP90s activities. Furthermore, HSP90s interacted with and destabilized ELF3. In the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) mutant, the changes in endogenous PIF4 and ELF3 protein levels caused by the inhibition of HSP90s activities were abolished. HSP90s enhanced the interaction between COP1 and ELF3, reduced ELF3 functional effects on PIF4 and modulated hypocotyl elongation during skoto- and thermomorphogenesis. Our results indicated that HSP90s participate in light and temperature signalling via the COP1-ELF3-PIF4 module to regulate hypocotyl growth in changing environments.

Maize WRKY28 interacts with the DELLA protein D8 to affect skotomorphogenesis and participates in the regulation of shade avoidance and plant architecture.

Competition for light from neighboring vegetation can trigger the shade-avoidance response (SAR) in plants, which is detrimental to their yield. The molecular mechanisms regulating SAR are well established in Arabidopsis, and some regulators of skotomorphogenesis have been found to be involved in the regulation of the SAR and plant architecture. However, the role of WRKY transcription factors in this process has rarely been reported, especially in maize (Zea mays). Here, we report that maize Zmwrky28 mutants exhibit shorter mesocotyls in etiolated seedlings. Molecular and biochemical analyses demonstrate that ZmWRKY28 directly binds to the promoter regions of the Small Auxin Up RNA (SAUR) gene ZmSAUR54 and the Phytochrome-Interacting Factor (PIF) gene ZmPIF4.1 to activate their expression. In addition, the maize DELLA protein Dwarf Plant8 (D8) interacts with ZmWRKY28 in the nucleus to inhibit its transcriptional activation activity. We also show that ZmWRKY28 participates in the regulation of the SAR, plant height, and leaf rolling and erectness in maize. Taken together, our results reveal that ZmWRKY28 is involved in GA-mediated skotomorphogenic development and can be used as a potential target to regulate SAR for breeding of high-density-tolerant cultivars.

Oxidative signalling in seed germination and early seedling growth: an emerging role for ROS trafficking and inter-organelle communication.

Underground early development of higher plants includes two distinct developmental processes, seed germination and then skotomorphogenesis, a mechanism which favours elongation of the hypocotyl and helps the seedling to find light. Interestingly, both processes, which are regulated by plant hormones, have been shown to depend on reactive oxygen species metabolism and to be related to mitochondrial retrograde signalling. Here we review the recent outcomes in this field of research and highlight the emerging role of ROS communication between organelles and cell compartments. We point out the role of mitochondria as an environmental and developmental sensor organelle that regulates ROS homeostasis and downstream events and we propose future directions of research that should help better understanding the roles of ROS in germination and seedling emergence.

Processing bodies control the selective translation for optimal development of Arabidopsis young seedlings.

Germinated plant seeds buried in soil undergo skotomorphogenic development before emergence to reach the light environment. Young seedlings transitioning from dark to light undergo photomorphogenic development. During photomorphogenesis, light alters the transcriptome and enhances the translation of thousands of mRNAs during the dark-to-light transition in Arabidopsis young seedlings. About 1,500 of these mRNAs have comparable abundance before and after light treatment, which implies widespread translational repression in dark-grown seedlings. Processing bodies (p-bodies), the cytoplasmic granules found in diverse organisms, can balance the storage, degradation, and translation of mRNAs. However, the function of p-bodies in translation control remains largely unknown in plants. Here we found that an Arabidopsis mutant defective in p-body formation (Decapping 5; dcp5-1) showed reduced fitness under both dark and light conditions. Comparative transcriptome and translatome analyses of wild-type and dcp5-1 seedlings revealed that p-bodies can attenuate the premature translation of specific mRNAs in the dark, including those encoding enzymes for protochlorophyllide synthesis and PIN-LIKES3 for auxin-dependent apical hook opening. When the seedlings protrude from soil, light perception by photoreceptors triggers a reduced accumulation of p-bodies to release the translationally stalled mRNAs for active translation of mRNAs encoding proteins needed for photomorphogenesis. Our data support a key role for p-bodies in translation repression, an essential mechanism for proper skotomorphogenesis and timely photomorphogenesis in seedlings.

Dual function of HYPONASTIC LEAVES 1 during early skotomorphogenic growth in Arabidopsis.

Seeds germinating underground display a specific developmental programme, termed skotomorphogenesis, to ensure survival of the emerging seedlings until they reach the light. They rapidly elongate the hypocotyl and maintain the cotyledons closed, forming a hook with the hypocotyl in order to protect apical meristematic cells from mechanical damage. Such crucial events for the fate of the seedling are tightly regulated and although some transcriptional regulators and phytohormones are known to be implicated in this regulation, we are still far from a complete understanding of these biological processes. Our work provides information on the diverse roles in skotomorphogenesis of the core components of microRNA biogenesis in Arabidopsis, HYL1, DCL1, and SE. We show that hypocotyl elongation is promoted by all these components, probably through the action of specific miRNAs. Hook development also depends on these proteins however, remarkably, HYL1 exerts its role in an opposite way to DCL1 and SE. Interestingly, we found that a specific HYL1 domain involved in protein-protein interaction is required for this function. Genetic evidences also point to the phosphorylation status of HYL1 as important for this function. We propose that HYL1 help maintain the hook closed during early skotomorphogenesis in a microprocessor-independent manner by repressing the activity of HY5, the transcriptional master regulator that triggers light responses. This work uncovers a previously unnoticed link between components of the miRNA biogenesis machinery, the skotomorphogenic growth, and hook development in Arabidopsis.

Chromatin enrichment for proteomics in plants (ChEP-P) implicates the histone reader ALFIN-LIKE 6 in jasmonate signalling.

BACKGROUND: Covalent modifications of core histones govern downstream DNA-templated processes such as transcription by altering chromatin structure and function. Previously, we reported that the plant homeodomain protein ALFIN-LIKE 6 (AL6), a bona fide histone reader that preferentially binds trimethylated lysin 4 on histone 3 (H3K4me3), is critical for recalibration of cellular phosphate (Pi) homeostasis and root hair elongation under Pi-deficient conditions. RESULTS: Here, we demonstrate that AL6 is also involved in the response of Arabidopsis seedlings to jasmonic acid (JA) during skotomorphogenesis, possibly by modulating chromatin dynamics that affect the transcriptional regulation of JA-responsive genes. Dark-grown al6 seedlings showed a compromised reduction in hypocotyl elongation upon exogenously supplied JA, a response that was calibrated by the availability of Pi in the growth medium. A comparison of protein profiles between wild-type and al6 mutant seedlings using a quantitative Chromatin Enrichment for Proteomics (ChEP) approach, that we modified for plant tissue and designated ChEP-P (ChEP in Plants), yielded a comprehensive suite of chromatin-associated proteins and candidates that may be causative for the mutant phenotype. CONCLUSIONS: Altered abundance of proteins involved in chromatin organization in al6 seedlings suggests a role of AL6 in coordinating the deposition of histone variants upon perception of internal or environmental stimuli. Our study shows that ChEP-P is well suited to gain holistic insights into chromatin-related processes in plants. Data are available via ProteomeXchange with identifier PXD026541.

Molecular bases for the constitutive photomorphogenic phenotypes in Arabidopsis.

The transition from skotomorphogenesis to photomorphogenesis is regulated in part by the COP1/SPA complex and phytochrome-interacting factors (PIFs) in Arabidopsis The constitutive photomorphogenic (cop) phenotypes of cop1 and spaQ mutants have been shown to result from a high abundance of positively acting transcription factors. Here, we show that the four major PIF proteins are unstable in cop1 mutants and that overexpression of PIF1, PIF3, PIF4 and PIF5 suppresses cop1 phenotypes in the dark. A comparison of the transcriptome data among cop1, spaQ and pifQ reveals remarkably overlapping gene expression profiles with preferential regulation of PIF direct target genes. Additionally, HFR1 strongly inhibits the in vivo binding and transcriptional activation activity of PIF1 in the dark. Taken together, these data suggest that the cop phenotypes of the cop1 and spaQ mutants are due to a combination of the reduced level of PIFs, increased levels of positively acting transcription factors (e.g. HY5/HFR1) and the HFR1-mediated inhibition of PIF-targeted gene expression in the dark. This article has an associated 'The people behind the papers' interview.

Integration of Light and Brassinosteroid Signaling during Seedling Establishment.

Light and brassinosteroid (BR) are external stimuli and internal cue respectively, that both play critical roles in a wide range of developmental and physiological process. Seedlings grown in the light exhibit photomorphogenesis, while BR promotes seedling etiolation. Light and BR oppositely control the development switch from shotomorphogenesis in the dark to photomorphogenesis in the light. Recent progress report that substantial components have been identified as hubs to integrate light and BR signals. Photomorphogenic repressors including COP1, PIFs, and AGB1 have been reported to elevate BR response, while photomorphogenesis-promoting factors such as HY5, BZS1, and NF-YCs have been proven to repress BR signal. In addition, BR components also modulate light signal. Here, we review the current research on signaling network associated with light and brassinosteroids, with a focus on the integration of light and BR signals enabling plants to thrive in the changeable environment.

Exogenous Application of Low-Concentration Sugar Enhances Brassinosteroid Signaling for Skotomorphogenesis by Promoting BIN2 Degradation.

In plants, seedling growth is subtly controlled by multiple environmental factors and endogenous phytohormones. The cross-talk between sugars and brassinosteroid (BR) signaling is known to regulate plant growth; however, the molecular mechanisms that coordinate hormone-dependent growth responses with exogenous sucrose in plants are incompletely understood. Skotomorphogenesis is a plant growth stage with rapid elongation of the hypocotyls. In the present study, we found that low-concentration sugars could improve skotomorphogenesis in a manner dependent on BR biosynthesis and TOR activation. However, accumulation of BZR1 in bzr1-1D mutant plants partially rescued the defects of skotomorphogenesis induced by the TOR inhibitor AZD, and these etiolated seedlings displayed a normal phenotype like that of wild-type seedlings in response to both sucrose and non-sucrose treatments, thereby indicating that accumulated BZR1 sustained, at least partially, the sucrose-promoted growth of etiolated seedlings (skotomorphogenesis). Moreover, genetic evidence based on a phenotypic analysis of bin2-3bil1bil2 triple-mutant and gain-of-function bin2-1 mutant plant indicated that BIN2 inactivation was conducive to skotomorphogenesis in the dark. Subsequent biochemical and molecular analyses enabled us to confirm that sucrose reduced BIN2 levels via the TOR-S6K2 pathway in etiolated seedlings. Combined with a determination of the cellulose content, our results indicated that sucrose-induced BIN2 degradation led to the accumulation of BZR1 and the enhancement of cellulose synthesis, thereby promoting skotomorphogenesis, and that BIN2 is the converging node that integrates sugar and BR signaling.

Beyond the darkness: recent lessons from etiolation and de-etiolation studies.

The state of etiolation is generally defined by the presence of non-green plastids (etioplasts) in plant tissues that would normally contain chloroplasts. In the commonly used dark-grown seedling system, etiolation is coupled with a type of growth called skotomorphogenesis. Upon illumination, de-etiolation occurs, marked by the transition from etioplast to chloroplast, and, at the seedling level, a switch to photomorphogenic growth. Etiolation and de-etiolation systems are therefore important for understanding both the acquisition of photosynthetic capacity during chloroplast biogenesis and plant responses to light-the most relevant signal in the life and growth of the organism. In this review, we discuss recent discoveries (within the past 2-3 years) in the field of etiolation and de-etiolation, with a particular focus on post-transcriptional processes and ultrastructural changes. We further discuss ambiguities in definitions of the term 'etiolation', and benefits and biases of common etiolation/de-etiolation systems. Finally, we raise several open questions and future research possibilities.

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin.

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.

The MED7 subunit paralogs of Mediator function redundantly in development of etiolated seedlings in Arabidopsis.

MED7 is a subunit of the Mediator middle module and is encoded by two paralogs in Arabidopsis. We generated MED7 silenced lines using RNAi to study its impact on Arabidopsis growth and development. Compared with wild type, etiolated seedlings of the MED7 silenced lines exhibited reduced hypocotyl length caused by reduced cell elongation when grown in the dark. The hypocotyl length phenotype was rescued by exogenously supplied brassinosteroid. In addition, MED7 silenced seedlings exhibited defective hook opening in the dark as well as defective cotyledon expansion in the presence of the brassinosteroid inhibitor brassinazole. Whole transcriptome analysis on etiolated seedlings using RNA sequencing revealed several genes known to be regulated by auxin and brassinosteroids, and a broad range of cell wall-related genes that were differentially expressed in the MED7 silenced lines. This was especially evident for genes involved in cell wall extension and remodeling, such as EXPANSINs and XTHs. Conditional complementation with each MED7 paralog individually restored the hypocotyl phenotype as well as the gene expression defects. Additionally, conditional expression of MED7 had no effects that were independent of the Mediator complex on the observed phenotypes. We concluded that the MED7 paralogs function redundantly in regulating genes required for the normal development of etiolated Arabidopsis seedlings.

AtCRK5 Protein Kinase Exhibits a Regulatory Role in Hypocotyl Hook Development during Skotomorphogenesis.

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.

Jasmonate inhibits COP1 activity to suppress hypocotyl elongation and promote cotyledon opening in etiolated Arabidopsis seedlings.

A germinating seedling undergoes skotomorphogenesis to emerge from the soil and reach for light. During this phase, the cotyledons are closed, and the hypocotyl elongates. Upon exposure to light, the seedling rapidly switches to photomorphogenesis by opening its cotyledons and suppressing hypocotyl elongation. The E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is critical for maintaining skotomorphogenesis. Here, we report that jasmonate (JA) suppresses hypocotyl elongation and stimulates cotyledon opening in etiolated seedlings, partially phenocopying cop1 mutants in the dark. We also find that JA stabilizes several COP1-targeted transcription factors in a COP1-dependent manner. RNA-seq analysis further defines a JA-light co-modulated and cop1-dependent transcriptome, which is enriched for auxin-responsive genes and genes participating in cell wall modification. JA suppresses COP1 activity through at least two distinct mechanisms: decreasing COP1 protein accumulation in the nucleus; and reducing the physical interaction between COP1 and its activator, SUPPRESSOR OF PHYTOCHROME A-105 1 (SPA1). Our work reveals that JA suppresses COP1 activity to stabilize COP1 targets, thereby inhibiting hypocotyl elongation and stimulating cotyledon unfolding in etiolated Arabidopsis seedlings.

Melatonin is involved in skotomorphogenesis by regulating brassinosteroid biosynthesis in rice plants.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis catalyzing the conversion of serotonin into N-acetylserotonin. In plants, SNAT is encoded by 2 isogenes of which SNAT1 is constitutively expressed and its overexpression confers increased yield in rice. However, the role of SNAT2 remains to be clarified. In contrast to SNAT1, the diurnal rhythm of SNAT2 mRNA expression peaks at night. In this study, transgenic rice plants in which SNAT2 expression were suppressed by RNAi technology showed a decrease in melatonin and a dwarf phenotype with erect leaves, reminiscent of brassinosteroids (BR)-deficient mutants. Of note, the dwarf phenotype was dependent on the presence of dark, suggesting that melatonin is involved in dark growth (skotomorphogenesis). In support of this suggestion, SNAT2 RNAi lines exhibited photomorphogenic phenotypes such as inhibition of internodes and increased expression of light-inducible CAB genes in the dark. The causative gene for the melatonin-mediated BR biosynthetic gene was DWARF4, a rate-limiting BR biosynthetic gene. Exogenous melatonin treatment induced several BR biosynthetic genes, including DWARF4, D11, and RAVL1. As expected from the erect leaves, the SNAT2 RNAi lines produced less BR than the wild type. Our results show for the first time that melatonin is a positive regulator of dark growth or shade outgrowth by regulating BR biosynthesis in plants.

Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana.

Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (responsive to desiccation 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles' heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP.

NADPH:protochlorophyllide oxidoreductase B (PORB) action in Arabidopsis thaliana revisited through transgenic expression of engineered barley PORB mutant proteins.

NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme for the light-induced greening of etiolated angiosperm plants. It belongs to the 'RED' family of reductases, epimerases and dehydrogenases. All POR proteins characterized so far contain evolutionarily conserved cysteine residues implicated in protochlorophyllide (Pchlide)-binding and catalysis. cDNAs were constructed by site-directed mutagenesis that encode PORB mutant proteins with defined Cys→Ala exchanges. These cDNAs were expressed in transgenic plants of a PORB-deficient knock-out mutant (porB) of Arabidopsis thaliana. Results show that porB plants expressing PORB mutant proteins with Ala substitutions of Cys276 or Cys303 are hypersensitive to high-light conditions during greening. Hereby, failure to assemble higher molecular weight complexes of PORB with its twin isoenzyme, PORA, as encountered with (Cys303→Ala)-PORB plants, caused more severe effects than replacing Cys276 by an Ala residue in the active site of the enzyme, as encountered in (Cys276→Ala)-PORB plants. Our results are consistent with the presence of two distinct pigment binding sites in PORB, with Cys276 establishing the active site of the enzyme and Cys303 providing a second, low affinity pigment binding site that is essential for the assembly of higher molecular mass light-harvesting PORB::PORA complexes and photoprotection of etiolated seedlings. Failure to assemble such complexes provoked photodynamic damage through the generation of singlet oxygen. Together, our data highlight the importance of PORB for Pchlide homoeostasis and greening in Arabidopsis.