Plant cuticles repress organ initiation and development during skotomorphogenesis in Arabidopsis.

After germination in the dark, plants produce a shoot apical hook and closed cotyledons to protect the quiescent shoot apical meristem (SAM), which is critical for seedling survival during skotomorphogenesis. The factors that coordinate these processes, particularly SAM repression, remain enigmatic. Plant cuticles, multilayered structures of lipid components on the outermost surface of the aerial epidermis of all land plants, provide protection against desiccation and external environmental stresses. Whether and how cuticles regulate plant development are still unclear. Here, we demonstrate that mutants of BODYGUARD1 (BDG1) and long-chain acyl-CoA synthetase2 (LACS2), key genes involved in cutin biosynthesis, produce a short hypocotyl with an opened apical hook and cotyledons in which the SAM is activated during skotomorphogenesis. Light signaling represses expression of BDG1 and LACS2, as well as cutin biosynthesis. Transcriptome analysis revealed that cuticles are critical for skotomorphogenesis, particularly for the development and function of chloroplasts. Genetic and molecular analyses showed that decreased HOOKLESS1 expression results in apical hook opening in the mutants. When hypoxia-induced expression of LITTLE ZIPPER2 at the SAM promotes organ initiation in the mutants, the de-repressed expression of cell-cycle genes and the cytokinin response induce the growth of true leaves. Our results reveal previously unrecognized developmental functions of the plant cuticle during skotomorphogenesis and demonstrate a mechanism by which light initiates photomorphogenesis through dynamic regulation of cuticle synthesis to induce coordinated and systemic changes in organ development and growth during the skotomorphogenesis-to-photomorphogenesis transition.

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.

Role of plastids and mitochondria in the early development of seedlings in dark growth conditions.

Establishment of the seedlings is a crucial stage of the plant life cycle. The success of this process is essential for the growth of the mature plant. In Nature, when seeds germinate under the soil, seedlings follow a dark-specific program called skotomorphogenesis, which is characterized by small, non-green cotyledons, long hypocotyl, and an apical hook-protecting meristematic cells. These developmental structures are required for the seedlings to emerge quickly and safely through the soil and gain autotrophy before the complete depletion of seed resources. Due to the lack of photosynthesis during this period, the seed nutrient stocks are the primary energy source for seedling development. The energy is provided by the bioenergetic organelles, mitochondria, and etioplast (plastid in the dark), to the cell in the form of ATP through mitochondrial respiration and etio-respiration processes, respectively. Recent studies suggest that the limitation of the plastidial or mitochondrial gene expression induces a drastic reprogramming of the seedling morphology in the dark. Here, we discuss the dark signaling mechanisms involved during a regular skotomorphogenesis and how the dysfunction of the bioenergetic organelles is perceived by the nucleus leading to developmental changes. We also describe the probable involvement of several plastid retrograde pathways and the interconnection between plastid and mitochondria during seedling development. Understanding the integration mechanisms of organellar signals in the developmental program of seedlings can be utilized in the future for better emergence of crops through the soil.

MORF2-mediated plastidial retrograde signaling is involved in stress response and skotomorphogenesis beyond RNA editing.

Retrograde signaling modulates the expression of nuclear genome-encoded organelle proteins to adjust organelle function in response to environmental cues. MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2) was initially recognized as a plastidial RNA-editing factor but recently shown to interact with GUN1. Given the central role of GUN1 in chloroplast retrograde signaling and the unviable phenotype of morf2 mutants that is inconsistent with many viable mutants involved in RNA editing, we hypothesized that MORF2 has functions either dosage dependent or beyond RNA editing. Using an inducible Clustered Interspaced Short Palindromic Repeat interference (iCRISPRi) approach, we were able to reduce the MORF2 transcripts in a controlled manner. In addition to MORF2-dosage dependent RNA-editing errors, we discovered that reducing MORF2 by iCRISPRi stimulated the expression of stress responsive genes, triggered plastidial retrograde signaling, repressed ethylene signaling and skotomorphogenesis, and increased accumulation of hydrogen peroxide. These findings along with previous discoveries suggest that MORF2 is an effective regulator involved in plastidial metabolic pathways whose reduction can readily activate multiple retrograde signaling molecules possibly involving reactive oxygen species to adjust plant growth. In addition, our newly developed iCRISPRi approach provided a novel genetic tool for quantitative reverse genetics studies on hub genes in plants.

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.

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.

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.

Metabolic network changes during skotomorphogenesis in Arabidopsis thaliana mutant (atdfb-3).

The metabolic networks underlying skotomorphogenesis in seedlings remain relatively unknown. On the basis of our previous study on the folate metabolism in seedlings grown in darkness, the plastidial folylpolyglutamate synthetase gene (AtDFB) T-DNA insertion Arabidopsis thaliana mutant (atdfb-3) was examined. Under the nitrate-sufficient condition, the mutant exhibited deficient folate metabolism and hypocotyl elongation, which affected skotomorphogenesis. Further analyses revealed changes to multiple intermediate metabolites related to carbon and nitrogen metabolism in the etiolated atdfb-3 seedlings. Specifically, the sugar, polyol, and fatty acid contents decreased in the atdfb-3 mutant under the nitrate-sufficient condition, whereas the abundance of various organic acids and amino acids increased. In response to nitrate-limited stress, multiple metabolites, including sugars, polyols, fatty acids, organic acids, and amino acids, accumulated more in the mutant than in the wild-type control. The differences in the contents of multiple metabolites between the atdfb-3 and wild-type seedlings decreased following the addition of exogenous 5-F-THF under both nitrogen conditions. Additionally, the mutant accumulated high levels of one-carbon metabolites, such as Cys, S-adenosylmethionine, and S-adenosylhomocysteine, under both nitrogen conditions. Thus, our data demonstrated that the perturbed folate metabolism in the atdfb-3 seedlings, which was caused by the loss-of-function mutation to AtDFB, probably altered carbon and nitrogen metabolism, thereby modulating skotomorphogenesis. Furthermore, the study findings provide new evidence of the links among folate metabolism, metabolic networks, and skotomorphogenesis.

MSD2, an apoplastic Mn-SOD, contributes to root skotomorphogenic growth by modulating ROS distribution in Arabidopsis.

Reactive oxygen species (ROS) play essential roles as a second messenger in various physiological processes in plants. Due to their oxidative nature, ROS can also be harmful. Thus, the generation and homeostasis of ROS are tightly controlled by multiple enzymes. Membrane-localized NADPH oxidases are well known to generate ROS during developmental and stress responses, but the metabolic pathways of the superoxide (O2-) generated by them in the apoplast are poorly understood, and the identity of the apoplastic superoxide dismutase (SOD) is unknown in Arabidopsis. Here, we show that a putative manganese SOD, MSD2 is secreted and possesses a SOD activity that can be inhibited by nitration at tyrosine 68. The expression of MSD2 in roots is light condition-dependent, suggesting that MSD2 may act on ROS metabolism in roots during the light-to-dark transition. Root architecture is governed by ROS distribution that exhibits opposite gradient of H2O2 and O2-, which is indeed altered in etiolated msd2 mutants and accompanied by changes in the onset of differentiation. These results provide a missing link in our understanding of ROS metabolism and suggest that MSD2 plays a role in root skotomorphogenesis by regulating ROS distribution, thereby playing a pivotal role in plant growth and development.

An improved repertoire of splicing variants and their potential roles in Arabidopsis photomorphogenic development.

BACKGROUND: Light switches on the photomorphogenic development of young plant seedlings, allowing young seedlings to acquire photosynthetic capacities and gain survival fitness. Light regulates gene expression at all levels of the central dogma, including alternative splicing (AS) during the photomorphogenic development. However, accurate determination of full-length (FL) splicing variants has been greatly hampered by short-read RNA sequencing technologies. RESULT: In this study, we adopt PacBio isoform sequencing (Iso-seq) to overcome the limitation of the short-read RNA-seq technologies. Normalized cDNA libraries used for Iso-seq allows for comprehensive and effective identification of FL AS variants. Our analyses reveal more than 30,000 splicing variant models from approximately 16,500 gene loci and additionally identify approximately 700 previously unannotated genes. Among the variants, approximately 12,000 represent new gene models. Intron retention (IR) is the most frequently observed form of variants, and many IR-containing AS variants show evidence of engagement in translation. Our study reveals the formation of heterodimers of transcription factors composed of annotated and IR-containing AS variants. Moreover, transgenic plants overexpressing the IR forms of two B-BOX DOMAIN PROTEINs exhibits light-hypersensitive phenotypes, suggesting their regulatory roles in modulating optimal light responses. CONCLUSIONS: This study provides an accurate and comprehensive portrait of full-length transcript isoforms and experimentally confirms the presence of de novo synthesized AS variants that impose regulatory functions in photomorphogenic development in Arabidopsis.

Comparative effects of polystyrene nanoplastics with different surface charge on seedling establishment of Chinese cabbage (Brassica rapa L.).

Micro- and nano-plastics are common emerging pollutants of great interest. However, the impacts of them on terrestrial plants were still poorly understood. In this study, comparative effects of exposure of polystyrene nanoplastics (PS) and amino-modified polystyrene nanoplastics (PS-NH2) on Chinese cabbage (Brassica rapa L.) plants at different growth stages were investigated. Hydroponically cultured seedlings were exposed to PS and PS-NH2 at 0, 1, 10, and 100 mg/L at skotomorphogenesis stage for 48 h, photomorphogenesis stage for 18 h, and the whole stage, respectively. Results showed that both PS and PS-NH2 had no discernible effect on radicle elongation at the skotomorphogenesis stage whereas significantly (P < 0.05) reduced photosynthetic pigment contents in varying degrees (18.06%-28.52%, 22.46%-36.86%) at the photomorphogenesis stage and the whole stage. Moreover, there was no significant difference between PS treatments and control except the 26.52% decline of chlorophyll a content at 1 mg/L at photomorphogenesis, while PS-NH2 significantly (P < 0.05) decreased photosynthetic pigment contents except the chlorophyll b content at 10 mg/L at photomorphogenesis. The content of chlorophyll a decreased by 26.68% for the PS-NH2-treated group and 22.46% for the PS-treated group at 1 mg/L during the whole stage. Results manifested that less negatively charged PS-NH2 seemed to show more severe phytotoxicity both at the photomorphogenesis stage and the whole stage. Notably, the surface charge of nano-plastics and the integrity of seedling establishment could be the main factors impacting the above difference. These findings are expected to improve our understanding of the effects of PSNPs on crop plants.

Role of Basal ABA in Plant Growth and Development.

Abscisic acid (ABA) regulates various aspects of plant physiology, including promoting seed dormancy and adaptive responses to abiotic and biotic stresses. In addition, ABA plays an im-portant role in growth and development under non-stressed conditions. This review summarizes phenotypes of ABA biosynthesis and signaling mutants to clarify the roles of basal ABA in growth and development. The promotive and inhibitive actions of ABA in growth are characterized by stunted and enhanced growth of ABA-deficient and insensitive mutants, respectively. Growth regulation by ABA is both promotive and inhibitive, depending on the context, such as concentrations, tissues, and environmental conditions. Basal ABA regulates local growth including hyponastic growth, skotomorphogenesis and lateral root growth. At the cellular level, basal ABA is essential for proper chloroplast biogenesis, central metabolism, and expression of cell-cycle genes. Basal ABA also regulates epidermis development in the shoot, by inhibiting stomatal development, and deposition of hydrophobic polymers like a cuticular wax layer covering the leaf surface. In the root, basal ABA is involved in xylem differentiation and suberization of the endodermis. Hormone crosstalk plays key roles in growth and developmental processes regulated by ABA. Phenotypes of ABA-deficient and insensitive mutants indicate prominent functions of basal ABA in plant growth and development.

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.

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.

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.

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.

Experimental Procedures for Studying Skotomorphogenesis in Arabidopsis thaliana.

Seedlings grown in darkness exhibit distinct morphologies comparing with light-grown seedlings. Elongated hypocotyls, closed yellow cotyledons, and the formation of apical hooks are typical characteristics for etiolated seedlings, which are collectively named skotomorphogenesis. Various plant hormones and environmental factors are essential for maintaining skotomorphogenesis. Due to the diverse morphological outcomes in etiolated seedlings grown under different treatments, studies on skotomorphogenesis are of particular importance to reveal the molecular mechanisms underlying plant response to environmental cues. Here, we detailed experimental procedures to facilitate researchers who are investigating etiolation growth-related studies.