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In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including β-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit β-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex. The starch hydrolysis activity of BAM1 drastically increased in the presence of LSF1-MDH in vitro. We determined the structure of the BAM1-LSF1-MDH complex by a combination of cryo-electron microscopy, crosslinking mass spectrometry, and molecular docking. The starch-binding domain of the dual-specificity phosphatase and carbohydrate-binding module of LSF1 was docked in proximity to BAM1, thus facilitating BAM1 access to and hydrolysis of the polyglucans of starch, thus revealing the molecular mechanism by which the LSF1-MDH complex improves the starch degradation activity of BAM1. Moreover, LSF1 is phosphatase inactive, and the enzymatic activity of MDH was dispensable for starch degradation, suggesting nonenzymatic scaffold functions for LSF1-MDH in starch degradation. These findings provide important insights into the precise regulation of starch degradation., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The plant UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) exists as a homodimer in its inactive ground state. Upon UV-B exposure, UVR8 monomerizes and interacts with a downstream key regulator, the CONSTITUTIVE PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA (COP1/SPA) E3 ubiquitin ligase complex, to initiate UV-B signaling. Two WD40 proteins, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 directly interact with monomeric UVR8 and facilitate UVR8 ground state reversion, completing the UVR8 photocycle. Here, we reconstituted the RUP-mediated UVR8 redimerization process in vitro and reported the structure of the RUP2-UVR8 W285A complex (2.0 Å). RUP2 and UVR8 W285A formed a heterodimer via two distinct interfaces, designated Interface 1 and 2. The previously characterized Interface 1 is found between the RUP2 WD40 domain and the UVR8 C27 subregion. The newly identified Interface 2 is formed through interactions between the RUP2 WD40 domain and the UVR8 core domain. Disruption of Interface 2 impaired UV-B induced photomorphogenic development in Arabidopsis thaliana. Further biochemical analysis indicated that both interfaces are important for RUP2-UVR8 interactions and RUP2-mediated facilitation of UVR8 redimerization. Our findings suggest that the two-interface-interaction mode is adopted by both RUP2 and COP1 when they interact with UVR8, marking a step forward in understanding the molecular basis that underpins the interactions between UVR8 and its photocycle regulators., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The blue-light receptor cryptochrome (CRY) in plants undergoes oligomerization to transduce blue-light signals after irradiation, but the corresponding molecular mechanism remains poorly understood. Here, we report the cryogenic electron microscopy structure of a blue-light-activated CRY2 tetramer at a resolution of 3.1 Å, which shows how the CRY2 tetramer assembles. Our study provides insights into blue-light-mediated activation of CRY2 and a theoretical basis for developing regulators of CRYs for optogenetic manipulation.

MS5 is a meiosis-related protein belonging to the Brassicaceae-specific domain of unknown function family and characterized by the MS5 superfamily domain (MSD). In this study, we elucidated the three-dimensional crystal structure and potential biochemical function of the MSD. It was observed that the MSD adopts a cystatin-like fold, mainly consisting of a central α-helix and four- or five-stranded antiparallel β-sheets that wrap around it. However, unlike cystatins, which inhibit cysteine proteases, the MSD displayed allosteric activation of papain. We believe that our study provides insight into novel mechanisms of proteolytic enzyme regulation and may serve as a basis for functional studies of the MS5 family proteins in plants., (© 2020 Federation of European Biochemical Societies.)

Abscisic acid (ABA) is a key phytohormone that mediates environmental stress responses. Vitamin C, or L-ascorbic acid (AsA), is the most abundant antioxidant protecting against stress damage in plants. How the ABA and AsA signaling pathways interact in stress responses remains elusive. In this study, we characterized the role of a previously unidentified gene, PTPN (PTP-like Nucleotidase) in plant drought tolerance. In Arabidopsis, (AtPTPN was expressed in multiple tissues and upregulated by ABA and drought treatments. Loss-of-function mutants of AtPTPN were hyposensitive to ABA but hypersensitive to drought stresses, whereas plants with enhanced expression of AtPTPN showed opposite phenotypes to . Overexpression of maize PTPN (ZmPTPN) promoted, while knockdown of ZmPTPN inhibited plant drought tolerance, indicating conserved and positive roles of PTPN in plant drought tolerance. We found that both AtPTPN and ZmPTPN release Pi by hydrolyzing GDP/GMP/dGMP/IMP/dIMP, and that AtPTPN positively regulated AsA production via endogenous Pi content control. Consistently, overexpression of VTC2, the rate-limiting synthetic enzyme in AsA biosynthesis, promoted AsA production and plant drought tolerance, and these effects were largely dependent on AtPTPN activity. Furthermore, we demonstrated that the heat shock transcription factor HSFA6a directly binds the AtPTPN promoter and activates AtPTPN expression. Genetic analyses showed that AtPTPN is required for HSFA6a to regulate ABA and drought responses. Taken together, our data indicate that PTPN-mediated crosstalk between the ABA signaling and AsA biosynthesis pathways positively controls plant drought tolerance., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

Cryptochromes (CRYs) are blue-light receptors in plants that harbor FAD as a cofactor and regulate various physiological responses. Photoactivated CRYs undergo oligomerization, which increases the binding affinity to downstream signaling partners. Despite decades of research on the activation of CRYs, little is known about how they are inactivated. Binding of blue-light inhibitors of cryptochromes (BICs) to CRY2 suppresses its photoactivation, but the underlying mechanism remains unknown. Here, we report crystal structures of CRY2N (CRY2 PHR domain) and the BIC2-CRY2N complex with resolutions of 2.7 and 2.5 Å, respectively. In the BIC2-CRY2N complex, BIC2 exhibits an extremely extended structure that sinuously winds around CRY2N. In this way, BIC2 not only restrains the transfer of electrons and protons from CRY2 to FAD during photoreduction but also interacts with the CRY2 oligomer to return it to the monomer form. Uncovering the mechanism of CRY2 inactivation lays a solid foundation for the investigation of cryptochrome protein function.

Members of the pentatricopeptide repeat (PPR) protein family are sequence-specific RNA-binding proteins that play crucial roles in organelle RNA metabolism. Each PPR protein consists of a tandem array of PPR motifs, each of which aligns to one nucleotide of the RNA target. The di-residues in the PPR motif, which are referred to as the PPR codes, determine nucleotide specificity. Numerous PPR codes are distributed among the vast number of PPR motifs, but the correlation between PPR codes and RNA bases is poorly understood, which hinders target RNA prediction and functional investigation of PPR proteins. To address this issue, we developed a modular assembly method for high-throughput construction of designer PPRs, and by using this method, 62 designer PPR proteins containing various PPR codes were assembled. Then, the correlation between these PPR codes and RNA bases was systematically explored and delineated. Based on this correlation, the web server PPRCODE (http://yinlab.hzau.edu.cn/pprcode) was developed. Our study will not only serve as a platform for facilitating target RNA prediction and functional investigation of the large number of PPR family proteins but also provide an alternative strategy for the assembly of custom PPRs that can potentially be used for plant organelle RNA manipulation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chloroplast biogenesis and development are highly complex processes requiring interaction between plastid and nuclear genomic products. Using a high-throughput screen for chloroplast biogenesis suppressors in Arabidopsis ( Arabidopsis thaliana ), we identified a suppressor of thf1 ( sot5 ) that displays virescent and serrated leaves. Further characterization revealed that sot5 mutants are defective in leaf adaxial and abaxial polarity and act as enhancers of asymmetric leaves2 Map-based cloning identified SOT5 as a gene previously named EMB2279 that encodes a plastid-targeted pentatricopeptide repeat (PPR) protein with 11 PPR motifs. A G-to-A mutation in sot5 leads to a significant decrease in splicing efficiency, generating two additional mRNA variants. As reported previously, the sot5 null mutation is embryo lethal. SOT5 is predicted to bind to specific RNA sequences found in plastid rpl2 and trnK genes, and we found decreased splicing efficiency of the rpl2 and trnK genes in sot5 mutants. Together, our results reveal that the PPR protein SOT5/EMB2279 is required for intron splicing of plastid rpl2 and trnK , providing insights into the role of plastid translation in the coupled development between chloroplasts and leaves., (© 2018 American Society of Plant Biologists. All rights reserved.)

Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)