PHYTOCHROME INTERACTING FACTOR Research Articles

Functions of Phytochrome Interacting Factors (PIFs) in Adapting Plants to Biotic and Abiotic Stresses.

Plants possess the remarkable ability to sense detrimental environmental stimuli and launch sophisticated signal cascades that culminate in tailored responses to facilitate their survival, and transcription factors (TFs) are closely involved in these processes. Phytochrome interacting factors (PIFs) are among these TFs and belong to the basic helix-loop-helix family. PIFs are initially identified and have now been well established as core regulators of phytochrome-associated pathways in response to the light signal in plants. However, a growing body of evidence has unraveled that PIFs also play a crucial role in adapting plants to various biological and environmental pressures. In this review, we summarize and highlight that PIFs function as a signal hub that integrates multiple environmental cues, including abiotic (i.e., drought, temperature, and salinity) and biotic stresses to optimize plant growth and development. PIFs not only function as transcription factors to reprogram the expression of related genes, but also interact with various factors to adapt plants to harsh environments. This review will contribute to understanding the multifaceted functions of PIFs in response to different stress conditions, which will shed light on efforts to further dissect the novel functions of PIFs, especially in adaption to detrimental environments for a better survival of plants.

PIF4 interacts with ABI4 to serve as a transcriptional activator complex to promote seed dormancy by enhancing ABA biosynthesis and signaling.

Transcriptional regulation plays a key role in the control of seed dormancy, and many transcription factors (TFs) have been documented. However, the mechanisms underlying the interactions between different TFs within a transcriptional complex regulating seed dormancy remain largely unknown. Here, we showed that TF PHYTOCHROME-INTERACTING FACTOR4 (PIF4) physically interacted with the abscisic acid (ABA) signaling responsive TF ABSCISIC ACID INSENSITIVE4 (ABI4) to act as a transcriptional complex to promote ABA biosynthesis and signaling, finally deepening primary seed dormancy. Both pif4 and abi4 single mutants exhibited a decreased primary seed dormancy phenotype, with a synergistic effect in the pif4/abi4 double mutant. PIF4 binds to ABI4 to form a heterodimer, and ABI4 stabilizes PIF4 at the protein level, whereas PIF4 does not affect the protein stabilization of ABI4. Subsequently, both TFs independently and synergistically promoted the expression of ABI4 and NCED6, a key gene for ABA anabolism. The genetic evidence is also consistent with the phenotypic, physiological and biochemical analysis results. Altogether, this study revealed a transcriptional regulatory cascade in which the PIF4-ABI4 transcriptional activator complex synergistically enhanced seed dormancy by facilitating ABA biosynthesis and signaling.

A warm temperature-released negative feedback loop fine-tunes PIF4-mediated thermomorphogenesis in Arabidopsis

Plants can sense temperature changes and adjust their growth accordingly. In Arabidopsis, high ambient temperatures stimulate stem elongation by activating a key thermoresponsive regulator, PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Here, we show that warmth promotes the nighttime transcription of GI, which is necessary for the high temperature-induced transcription of TOC1. Genetic analyses suggest that GI prevents excessive thermoresponsive growth by inhibiting PIF4, with this regulatory mechanism being partially reliant on TOC1. GI transcription is repressed by ELF3 and HY5, which concurrently inhibit PIF4 expression and activity. Temperature elevation causes the deactivation or degradation of ELF3 and HY5, leading to PIF4 activation and relief of GI transcriptional repression at high temperatures. This allows PIF4 to further activate GI transcription in response to elevated temperatures. GI, in turn, inhibits PIF4, establishing a negative feedback loop that fine-tunes PIF4 activity. In addition, we demonstrate that ELF3, HY5, and PIF4 regulate GI transcription by modulating the enrichment of histone variant H2A.Z at the GI locus. Together, our findings suggest that thermal release of a negative feedback loop finely adjusts plant thermomorphogenesis.

Molecular mechanisms underlying coordinated responses of plants to shade and environmental stresses.

Shade avoidance syndrome (SAS) is triggered by a low ratio of red (R) to far-red (FR) light (R/FR ratio), which is caused by neighbor detection and/or canopy shade. In order to compete for the limited light, plants elongate hypocotyls and petioles by deactivating phytochrome B (phyB), a major R light photoreceptor, thus releasing its inhibition of the growth-promoting transcription factors PHYTOCHROME-INTERACTING FACTORs. Under natural conditions, plants must cope with abiotic stresses such as drought, soil salinity, and extreme temperatures, and biotic stresses such as pathogens and pests. Plants have evolved sophisticated mechanisms to simultaneously deal with multiple environmental stresses. In this review, we will summarize recent major advances in our understanding of how plants coordinately respond to shade and environmental stresses, and will also discuss the important questions for future research. A deep understanding of how plants synergistically respond to shade together with abiotic and biotic stresses will facilitate the design and breeding of new crop varieties with enhanced tolerance to high-density planting and environmental stresses.

The Roles of Circadian Clock Genes in Plant Temperature Stress Responses.

Plants monitor day length and memorize changes in temperature signals throughout the day, creating circadian rhythms that support the timely control of physiological and metabolic processes. The DEHYDRATION-RESPONSE ELEMENT-BINDING PROTEIN 1/C-REPEAT BINDING FACTOR (DREB1/CBF) transcription factors are known as master regulators for the acquisition of cold stress tolerance, whereas PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is involved in plant adaptation to heat stress through thermomorphogenesis. Recent studies have shown that circadian clock genes control plant responses to temperature. Temperature-responsive transcriptomes show a diurnal cycle and peak expression levels at specific times of throughout the day. Circadian clock genes play essential roles in allowing plants to maintain homeostasis by accommodating temperature changes within the normal temperature range or by altering protein properties and morphogenesis at the cellular level for plant survival and growth under temperature stress conditions. Recent studies revealed that the central oscillator genes CIRCADIAN CLOCK ASSOCIATED 1/LATE ELONGATED HYPOCOTYL (CCA1/LHY) and PSEUDO-RESPONSE REGULATOR5/7/9 (PRR5/7/9), as well as the EVENING COMPLEX (EC) genes REVEILLE4/REVEILLE8 (REV4/REV8), were involved in the DREB1 pathway of the cold signaling transcription factor and regulated the thermomorphogenesis gene PIF4. Further studies showed that another central oscillator, TIMING OF CAB EXPRESSION 1 (TOC1), and the regulatory protein ZEITLUPE (ZTL) are also involved. These studies led to attempts to utilize circadian clock genes for the acquisition of temperature-stress resistance in crops. In this review, we highlight circadian rhythm regulation and the clock genes involved in plant responses to temperature changes, as well as strategies for plant survival in a rapidly changing global climate.

Genome-wide identification and comprehensive analysis of the phytochrome-interacting factor (PIF) gene family in wheat.

Phytochrome-interacting factors (PIFs) are essential transcription factors for plant growth, development, and stress responses. Although PIF genes have been extensively studied in many plant species, they have not been thoroughly investigated in wheat. Here, we identified 18 PIF genes in cultivated hexaploid wheat (Triticum aestivum L). Phylogenetic analysis, exon-intron structures, and motif compositions revealed the presence of four distinct groups of TaPIFs. Genome-wide collinearity analysis of PIF genes revealed the evolutionary history of PIFs in wheat, Oryza sativa, and Brachypodium distachyon. Cis-regulatory element analysis suggested that TaPIF genes indicated participated in plant development and stress responses. Subcellular localization assays indicated that TaPIF2-1B and TaPIF4-5B were transcriptionally active. Both were found to be localized to the nucleus. Gene expression analyses demonstrated that TaPIFs were primarily expressed in the leaves and were induced by various biotic and abiotic stresses and phytohormone treatments. This study provides new insights into PIF-mediated stress responses and lays a strong foundation for future investigation of PIF genes in wheat.

Rice PIFs: Critical regulators in rice development and stress response.

Phytochrome-interacting factors (PIFs) belong to a subfamily of the basic helix-loop-helix (bHLH) family of transcription factors, which serve as a "hub" for development and growth of plants. They have the capability to regulate the expression of many downstream genes, integrate multiple signaling pathways, and act as a signaling center within the cell. In rice (Oryza sativa), the PIF family genes, known as OsPILs, play a crucial part in many different aspects. OsPILs play a crucial role in regulating various aspects of photomorphogenesis, skotomorphogenesis, plant growth, and development in rice. These vital processes include chlorophyll synthesis, plant gravitropism, plant height, flowering, and response to abiotic stress factors such as low temperature, drought, and high salt. Additionally, OsPILs are involved in controlling several important agronomic traits in rice. Some OsPILs members coordinate with each other to function. This review summarizes and prospects the latest research progress on the biological functions of OsPILs transcription factors and provides a reference for further exploring the functions and mechanism of OsPILs.

Heat Shock Factor A1s are required for phytochrome-interacting factor 4-mediated thermomorphogenesis in Arabidopsis.

Thermomorphogenesis and the heat shock (HS) response are distinct thermal responses in plants that are regulated by PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and HEAT SHOCK FACTOR A1s (HSFA1s), respectively. Little is known about whether these responses are interconnected and whether they are activated by similar mechanisms. An analysis of transcriptome dynamics in response to warm temperature (28°C) treatment revealed that 30 min of exposure activated the expression of a subset of HSFA1 target genes in Arabidopsis thaliana. Meanwhile, a loss-of-function HSFA1 quadruple mutant (hsfa1-cq) was insensitive to warm temperature-induced hypocotyl growth. In hsfa1-cq plants grown at 28°C, the protein and transcript levels of PIF4 were greatly reduced, and the circadian rhythm of many thermomorphogenesis-related genes (including PIF4) was disturbed. Additionally, the nuclear localization of HSFA1s and the binding of HSFA1d to the PIF4 promoter increased following warm temperature exposure, whereas PIF4 overexpression in hsfa1-cq partially rescued the altered warm temperature-induced hypocotyl growth of the mutant. Taken together, these results suggest that HSFA1s are required for PIF4 accumulation at a warm temperature, and they establish a central role for HSFA1s in regulating both thermomorphogenesis and HS responses in Arabidopsis.

GST Pull-Down Assay to Study PIF4 Binding In Vitro.

The phytochrome-interacting factor 4 (PIF4) is a well-known transcription factor that plays a pivotal role in plant thermomorphogenesis, coordinating growth and development in response to temperature changes. As PIF4 functions by forming complexes with other proteins, determining its interacting partners is essential for understanding its diverse roles in plant thermal responses. The GST (glutathione-S-transferase) pull-down assay is a widely used biochemical technique that enables the investigation of protein-protein interactions in vitro. It is particularly useful for studying transient or weak interactions between proteins. In this chapter, we describe the GST pull-down approach to detect the interaction between PIF4 and a known or suspected interacting protein. We provide detailed step-by-step descriptions of the assay procedures, from the preparation of recombinant GST-PIF4 fusion protein to the binding and elution of interacting partners. Additionally, we provide guidelines for data interpretation, quantification, and statistical analysis to ensure robust and reliable results.

Characterization of PIF4 Phosphorylation by SPA1.

The PHYTOCHROME INTERACTING FACTORs (PIFs) play pivotal roles in regulating thermo- and photo-morphogenesis in Arabidopsis. One of the main hubs in thermomorphogenesis is PIF4, which regulates plant development under high ambient temperature along with other PIFs. PIF4 enhances its own transcription and PIF4 protein is stabilized under high ambient temperature. However, the mechanisms of thermo-stabilization of PIF4 are less understood. Recently, it was shown that SUPPRESSOR OF PHYA-105 1 (SPA1) can function as a serine/threonine kinase to phosphorylate PIF4 in vitro, and the phosphorylated form of PIF4 is more stable under high ambient temperature conditions. In this chapter, we describe the in vitro kinase assay of PIF4 by SPA1. In principle, this protocol can be applied for other putative substrates and kinases.

Exogenous application of nanocarrier-mediated double-stranded RNA manipulates physiological traits and defence response against bacterial diseases.

Stability and delivery are major challenges associated with exogenous double-stranded RNA (dsRNA) application into plants. We report the encapsulation and delivery of dsRNA in cationic poly-aspartic acid-derived polymer (CPP6) into plant cells. CPP6 stabilizes the dsRNAs during long exposure at varied temperatures and pH, and protects against RNase A degradation. CPP6 helps dsRNA uptake through roots or foliar spray and facilitates systemic movement to induce endogenous gene silencing. The fluorescence of Arabidopsis GFP-overexpressing transgenic plants was significantly reduced after infiltration with gfp-dsRNA-CPP6 by silencing of the transgene compared to plants treated only with gfp-dsRNA. The plant endogenous genes flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) were downregulated by a foliar spray of ft-dsRNA-CPP6 and pif4-dsRNA-CPP6 in Arabidopsis, with delayed flowering and enhanced biomass. The rice PDS gene targeted by pds-dsRNA-CPP6 through root uptake was effectively silenced and plants showed a dwarf and albino phenotype. The NaCl-induced OsbZIP23 was targeted through root uptake of bzip23-dsRNA-CPP6 and showed reduced transcripts and seedling growth compared to treatment with naked dsRNA. The negative regulators of plant defence SDIR1 and SWEET14 were targeted through foliar spray to provide durable resistance against bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Overall, the study demonstrates that transient silencing of plant endogenous genes using polymer-encapsulated dsRNA provides prolonged and durable resistance against Xoo, which could be a promising tool for crop protection and for sustaining productivity.

Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

Transcription factors RhPIF4/8 and RhHY5 regulate autophagy-mediated petal senescence in rose (Rosa hybrida)

Petal senescence refers to the progressive loss of intracellular structures and functions within plant decorative organs, ultimately leading to cell death. Autophagy involves the degradation of damaged cellular components and nutrient recycling. Plant organ senescence and autophagy are highly coordinated; however, the mechanisms by which autophagy regulates petal senescence remain largely unknown. In this study, by using transmission electron microscopy, we observed that autophagic activity peaked early, at flower opening, without any senescence and other morphological symptoms in petals. We found that darkness positively regulated petal senescence and upregulated autophagy-related genes (ATGs). Dark treatment promoted the accumulation of Rosa hybrida phytochrome-interacting factor 4 (RhPIF4) in petals. RhPIF4 silencing delayed petal senescence and repressed the expression of ATGs. In contrast, silencing of the light-responsive gene Rosa hybridaelongated hypoctyl 5 (RhHY5) promoted petal senescence and ATG gene expression. RhPIF4/8 and RhHY5 could directly interact with RhWRKY40, and RhWRKY40 is directly bound to the promoters of RhATG7 and RhATG11. Silencing RhWRKY40 delayed petal senescence and suppressed RhATG7 and RhATG11 expression. Based on these results, we propose that RhPIF4/8 and RhHY5 transcription factors are involved in regulating petal senescence in response to dark or light conditions by modulating autophagic activity.

SPL16 and SPL23 mediate photoperiodic control of seasonal growth in Populus trees.

Perennial trees in boreal and temperate regions undergo growth cessation and bud set under short photoperiods, which are regulated by phytochrome B (phyB) photoreceptors and PHYTOCHROME INTERACTING FACTOR 8 (PIF8) proteins. However, the direct signaling components downstream of the phyB-PIF8 module remain unclear. We found that short photoperiods suppressed the expression of miR156, while upregulated the expression of miR156-targeted SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE 16 (SPL16) and SPL23 in leaves and shoot apices of Populus trees. Accordingly, either overexpression of MIR156a/c or mutagenesis of SPL16/23 resulted in the attenuation of growth cessation and bud set under short days (SD), whereas overexpression of SPL16 and SPL23 conferred early growth cessation. We further showed that SPL16 and SPL23 directly suppressed FLOWERING LOCUS T2 (FT2) expression while promoted BRANCHED1 (BRC1.1 and BRC1.2) expression. Moreover, we revealed that PIF8.1/8.2, positive regulators of growth cessation, directly bound to promoters of MIR156a and MIR156c and inhibited their expression to modulate downstream pathways. Our results reveal a connection between the phyB-PIF8 module-mediated photoperiod perception and the miR156-SPL16/23-FT2/BRC1 regulatory cascades in SD-induced growth cessation. Our study provides insights into the rewiring of a conserved miR156-SPL module in the regulation of seasonal growth in Populus trees.

Inhibition of demethylase by IOX1 modulates chromatin accessibility to enhance NSCLC radiation sensitivity through attenuated PIF1

Chromatin accessibility is a critical determinant of gene transcriptional expression and regulated by histones modification. However, the potential for manipulating chromatin accessibility to regulate radiation sensitivity remains unclear. Our findings demonstrated that the histone demethylase inhibitor, 5-carboxy-8-hydroxyquinoline (IOX1), could enhance the radiosensitivity of non-small cell lung cancer (NSCLC) in vitro and in vivo. Mechanistically, IOX1 treatment reduced chromatin accessibility in the promoter region of DNA damage repair genes, leading to decreased DNA repair efficiency and elevated DNA damage induced by γ irradiation. Notably, IOX1 treatment significantly reduced both chromatin accessibility and the transcription of phytochrome interacting factor 1 (PIF1), a key player in telomere maintenance. Inhibition of PIF1 delayed radiation-induced DNA and telomeric DNA damage repair, as well as increased radiosensitivity of NSCLC in vitro and in vivo. Further study indicated that the above process was regulated by a reduction of transcription factor myc-associated zinc finger protein (MAZ) binding to the distal intergenic region of the PIF1. Taken together, IOX1-mediated demethylase inactivation reduced chromatin accessibility, leading to elevated telomere damage which is partly due to PIF1 inhibition, thereby enhancing NSCLC radiosensitivity.

The interaction between CmPIF8 and CmACO1 under postharvest red light treatment might affect fruit ripening and sucrose accumulation in oriental melon fruit

Light and plant hormones are involved in regulating the growth, development and fruit quality formation. However, it remains unclear whether light regulates the fruit quality of oriental melon by affecting hormones. Sucrose is mainly accumulated in mature oriental melon fruit, and the accumulation of sucrose is closely related to ethylene. Here, the climacteric melon fruit ‘HS’ were treated with red and blue light at ethylene release initial stage (30 d after anthesis, DAA), and the results showed that the red light treatment enhanced the production of ethylene and the activity of 1-aminocyclopropane-1-carboxylate oxidase (ACO), as well as CmACO1 expression in melon fruit at postharvest stage. Red light also up-regulated the sucrose accumulation and the activity of sucrose phosphate synthetase (SPS) and CmSPS1 expression in postharvest melon fruit. Moreover, we found that PHYTOCHROME INTERACTING FACTOR 8 (PIF8), as a negative regulator of ethylene biosynthesis by directly binding to CmACO1 promoter. In summary, red light treatment at postharvest stage suppressed the expression of CmPIF8, and released the inhibitory effect of CmPIF8 on CmACO1, thereby promoting the ethylene production and fruit ripening, which indirectly increased the sucrose content in melon fruit.

Overexpression of GmPIF4b affects morpho-physiological traits to reduce heat-induced grain loss in soybean

Heat waves associated with climate change seriously threaten crop productivity. Crop seed yield depends on the success of reproduction. However, reproductive development is most vulnerable to heat stress conditions. Perception of heat and its conversion into cellular signals is a complex process. The basic helix loop helix (bHLH) transcription factor, Phytochrome Interacting Factor 4 (PIF4), plays a significant role in this process. However, studies on PIF4- mediated impacts on crop grain yield at a higher temperature are lacking. We investigated the overexpression of GmPIF4b in soybean to alleviate heat-induced damage and yield using a transgenic approach. Our results showed that under high-temperature conditions (38°C/28°C), overexpressing soybeans plants had higher chlorophyll a and b, and lower proline accumulation compared to WT. Further, overexpression of GmPIF4b improved pollen viability under heat stress and reduced heat-induced structural abnormalities in the male and female reproductive organs. Consequently, the transgenic plants produced higher pods and seeds per plant at high temperatures. Quantitative RT-PCR analysis showed that the overexpressing GmPIF4b soybeans had higher transcripts of heat shock factor, GmHSF-34, and heat-shock protein, GmHSP90A2. Collectively, our results suggest that GmPIF4b regulates multiple morpho-physiological traits for better yield under warmer climatic conditions.

Cover Image:

The BAP module, comprising BRASSINAZOLE RESISTANT 1 (BZR1), AUXIN RESPONSE FACTOR 6 (ARF6), and PHYTOCHROME‐INTERACTING FACTOR 4 (PIF4), is an important hub in plant signaling networks. Diao et al. (pages 2631‐2644) determined that the prototype BAP module arose in Marchantia polymorpha, underwent stepwise evolution and became established as a mature regulatory system in seed plants. This work elucidates the evolutionary trajectory of the BAP module and provides insight into the mechanisms by which plants to integrate environmental and endogenous signals. The cover image depicts the key players in the BAP module in bryophytes and seed plants.

Phytochrome B inhibits the activity of phytochrome-interacting factor 7 involving phase separation

Shade-intolerant plants sense changes in the light environment and trigger shade-avoidance syndrome in the presence of neighboring vegetation. Phytochrome-interacting factor 7 (PIF7) is an essential regulator that integrates shade signals into plant transcriptional networks. While the regulation of PIF7 under shade conditions has been well studied, the mechanism that represses PIF7 activity under white light remains ambiguous. Here, we report that PIF7 forms nuclear puncta containing phase-separated liquid-like condensates. Phytochrome B (phyB) then binds to dephosphorylated PIF7 and promotes its condensed phase of PIF7 under white light. The phyB-PIF7 condensate subsequently inhibits the DNA-binding activity of PIF7. However, shade inactivation of phyB causes the dissociation of phyB-PIF7 condensates and allows unbound PIF7 to promote the transcription of shade-induced genes. This reversible transcriptional condensation via phase separation provides sessile organisms with the flexibility of gene control to adapt to their surrounding environment.

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 β-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.