Vascular cambium produces cells to form xylem, or wood, in tree stems. Here, we explored transregulatory pathways in this cell lineage development. We tested 20 of the 95 putative vascular cambium-specific (VCS) transcription factor genes through gain-of-function transgenesis in Black cottonwood (Populus trichocarpa) and found that a SHORT-ROOT (PtrSHR1) gene induced some of the most prominent phenotypes. PtrSHR1 transcripts are in the vascular cambium (VC) but not in the stem-differentiating xylem (SDX), whereas PtrSHR1 proteins are in both VC and SDX. Constitutive overexpression or endonuclease-deficient Cas9 (dCas9)-mediated loci-specific gene activation of PtrSHR1 revealed an activated PtrSHR1-PtrWRKY25-PtrVCS2-PtrWOX4a/b (WUSCHEL-related homeobox 4a/b) regulatory path for an abbreviated VC zone and a smaller stem diameter. CRISPR PtrSHR1 mutation reduced the PtrSHR1-PtrWRKY25-PtrVCS2-PtrWOX4a/b regulation resulting in an expanded VC zone and a larger stem diameter. In SDX, PtrSHR1-PtrWRKY94 activated specifically 3 (Ptr4CL3, 4-coumarate:CoA ligase 3; PtrC3H3, p-coumarate 3-hydroxylase 3; and PtrC4H1, cinnamate 4-hydroxylase 1) of the 22 monolignol biosynthetic pathway genes to control lignin content and structure, corroborated by chemical degradation and 2-dimensional (2D) nuclear magnetic resonance analyses. Thus, PtrSHR1 mediates the proliferation of VC and diffuses into SDX to control lignin properties and wood production, unveiling potential strategies for creating advantageous wood feedstock for materials and energy.

A small genome in chloroplasts encodes many of the polypeptide subunits of the photosynthetic electron transport complexes embedded in the membranes of thylakoid vesicles in the chloroplast stroma and synthesized by ribosomes of the bacterial-like genetic system of this semiautonomous organelle. While thylakoid membranes (TMs) are sites of translation, evidence in the unicellular alga Chlamydomonas reinhardtii supports translation on noncanonical membranes in a discrete translation zone in the chloroplast. To characterize the membranous platforms for translation and the biogenesis of TMs, we profiled membranes during chloroplast development, using the yellow-in-the-dark1 mutant, and carried out proteomic analyses on 2 membrane types proposed previously to support translation in the chloroplast of C. reinhardtii: "low-density membrane" (LDM) and "chloroplast translation membrane" (CTM). The results support the roles of LDM and CTM in the preliminary and ongoing stages of translation, respectively. Proteomics, immunoprecipitation, and transmission electron microscopy results support connections of these membranous platforms and a chloroplast envelope domain bound by cytoplasmic ribosomes. Our results contribute to a model of photosynthesis complex biogenesis in a spatiotemporal "assembly line" involving LDM and CTM as sequential stages leading to photosynthetic TMs.

IntroductionVascular cambium proliferates and differentiates into the secondary xylem (wood), enabling the perennial increase in stem diameter for wood formation. In our previous study, we identified 95 vascular-cambium-specific (VCS) transcription factors (TFs) in Populus trichocarpa.MethodsIn this study, we characterized the function of the highly vascular cambium-expressed heat shock TF among these VCSs, PtrSCZ1, using PtrSCZ1-overexpressing transgenic lines and gene-edited mutants in P. trichocarpa.ResultsOverexpressing PtrSCZ1 or its homolog PtrSCZ3 (OE-PtrSCZ1, OE-PtrSCZ3) led to enhanced cambium activity, increased stem diameter, and a larger xylem proportion. CRISPR-based mutants of PtrSCZ1 and PtrSCZ3 exhibited phenotypes opposite to the OE-PtrSCZ1 and OE-PtrSCZ3 plants. This suggests that PtrSCZ1 and PtrSCZ3 redundantly promote cambium activity and secondary growth, leading to increased radial growth in P. trichocarpa. Overexpression and knockout of PtrSCZ1 and PtrSCZ3 significantly affected the expression of key regulatory factors of cambium (PtrWOX4a, PtrWOX4b, PtrWOX13a, PtrPXYa, PtrVCM1, and PtrVCM2) and disrupted cell wall-related gene expression. This demonstrates that PtrSCZ1 and PtrSCZ3 may function in cambium division activity by regulating these key cambium-associated transcription factors for wood formation.DiscussionOur work identifies PtrSCZ1 and PtrSCZ3 as promising target genes for enhancing wood yield through molecular breeding, and illustrates the role of vascular cambium systems in understanding lateral meristem development.

The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of Rubisco. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes. The expression of chloroplast proteins occurs in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

Blockchains can provide integrity and authenticity, but their limited storage capacity can be a challenge when it comes to storing large amounts of data. To address this issue, off-chain storage solutions such as the InterPlanetary File System (IPFS) can be utilized. This has led to the emergence of various applications that utilize both blockchain and IPFS. After reviewing a large body of literature utilizing blockchain and IPFS, we found that the coordinated interaction between blockchain and IPFS can help solve many problems and provide many research opportunities. Therefore, this survey paper aims to introduce the interaction mechanism between blockchain and IPFS. We first provide a general overview and comparison of different P2P data networks to help understand why IPFS is suitable as the storage layer for blockchains. Subsequently, we use select applications that leverage blockchain and IPFS to show how the mechanism works and explore new developments in this area. Specifically, we identify research areas and provide a qualitative comparison of these different applications. From the comparison, we derive research goals related to the interaction mechanism between blockchain and IPFS.

Summary Histone H3 lysine‐4 trimethylation (H3K4me3) activating drought‐responsive genes in plants for drought adaptation has long been established, but the underlying regulatory mechanisms are unknown. Here, using yeast two‐hybrid, bimolecular fluorescence complementation, biochemical analyses, transient and CRISPR‐mediated transgenesis in Populus trichocarpa , we unveiled in this adaptation a regulatory interplay between chromatin regulation and gene transactivation mediated by an epigenetic determinant, a PtrSDG2‐1–PtrCOMPASS (complex proteins associated with Set1)‐like H3K4me3 complex, PtrSDG2‐1–PtrWDR5a‐1–PtrRbBP5‐1–PtrAsh2‐2 (PtrSWRA). Under drought conditions, a transcription factor PtrAREB1‐2 interacts with PtrSWRA, forming a PtrSWRA–PtrAREB1‐2 pentamer, to recruit PtrSWRA to specific promoter elements of drought‐tolerant genes, such as PtrHox2 , PtrHox46 , and PtrHox52 , for depositing H3K4me3 to promote and maintain activated state of such genes for tolerance. CRISPR‐edited defects in the pentamer impaired drought tolerance and elevated expression of PtrHox2 , PtrHox46 , or PtrHox52 improved the tolerance as well as growth in P . trichocarpa . Our findings revealed the identity of the underlying H3K4 trimethyltransferase and its interactive arrangement with the COMPASS for catalysis specificity and efficiency. Furthermore, our study uncovered how the H3K4 trimethyltransferase–COMPASS complex is recruited to the effector genes for elevating H3K4me3 marks for improved drought tolerance and growth/biomass production in plants.

Chloroplasts are the defining plant organelles with responsibility for photosynthesis and other vital functions. To deliver these functions, they possess a complex proteome comprising thousands of largely nucleus-encoded proteins. Composition of the proteome is controlled by diverse processes affecting protein translocation and degradation-our focus here. Most chloroplast proteins are imported from the cytosol via multiprotein translocons in the outer and inner envelope membranes (the TOC and TIC complexes, respectively), or via one of several noncanonical pathways, and then sorted by different systems to organellar subcompartments. Chloroplast proteolysis is equally complex, involving the concerted action of internal proteases of prokaryotic origin and the nucleocytosolic ubiquitin-proteasome system (UPS). The UPS degrades unimported proteins in the cytosol and chloroplast-resident proteins via chloroplast-associated protein degradation (CHLORAD). The latter targets the TOC apparatus to regulate protein import, as well as numerous internal proteins directly, to reconfigure chloroplast functions in response to developmental and environmental signals.

Photosynthesis is the energetic basis for most life on Earth, and in plants it operates inside double membrane-bound organelles called chloroplasts. The photosynthetic apparatus comprises numerous proteins encoded by the nuclear and organellar genomes. Maintenance of this apparatus requires the action of internal chloroplast proteases, but a role for the nucleocytosolic ubiquitin-proteasome system (UPS) was not expected, owing to the barrier presented by the double-membrane envelope. Here, we show that photosynthesis proteins (including those encoded internally by chloroplast genes) are ubiquitinated and processed via the CHLORAD pathway: They are degraded by the 26S proteasome following CDC48-dependent retrotranslocation to the cytosol. This demonstrates that the reach of the UPS extends to the interior of endosymbiotically derived chloroplasts, where it acts to regulate photosynthesis, arguably the most fundamental process of life.

Tension wood (TW) is a specialized xylem tissue developed under mechanical/tension stress in angiosperm trees. TW development involves transregulation of secondary cell wall genes, which leads to altered wood properties for stress adaptation. We induced TW in the stems of black cottonwood (Populus trichocarpa, Nisqually-1) and identified two significantly repressed transcription factor (TF) genes: class B3 heat-shock TF (HSFB3-1) and MYB092. Transcriptomic analysis and chromatin immunoprecipitation (ChIP) were used to identify direct TF-DNA interactions in P. trichocarpa xylem protoplasts overexpressing the TFs. This analysis established a transcriptional regulatory network in which PtrHSFB3-1 and PtrMYB092 directly activate 8 and 11 monolignol genes, respectively. The TF-DNA interactions were verified for their specificity and transactivator roles in 35 independent CRISPR-based biallelic mutants and overexpression transgenic lines of PtrHSFB3-1 and PtrMYB092 in P. trichocarpa. The gene-edited trees (mimicking the repressed PtrHSFB3-1 and PtrMYB092 under tension stress) have stem wood composition resembling that of TW during normal growth and under tension stress (i.e., low lignin and high cellulose), whereas the overexpressors showed an opposite effect (high lignin and low cellulose). Individual overexpression of the TFs impeded lignin reduction under tension stress and restored high levels of lignin biosynthesis in the TW. This study offers biological insights to further uncover how metabolism, growth, and stress adaptation are coordinately regulated in trees.

Translation is localized within cells to target proteins to their proper locations. We asked whether translation occurs on the chloroplast surface in Chlamydomonas and, if so, whether it is involved in co-translational protein targeting, aligned spatially with localized translation by the bacterial-type ribosomes within this organelle, or both. Our results reveal a domain of the chloroplast envelope which is bound by translating ribosomes. Purified chloroplasts retained ribosomes and mRNAs encoding two chloroplast proteins specifically on this “translation domain”, but not a mRNA encoding a cytoplasmic protein. Ribosomes clusters were seen on this domain by electron tomography. Activity of the chloroplast-bound ribosomes is supported by results of the ribopuromycylation and puromycin-release assays. Co-translational chloroplast protein import is supported by nascent polypeptide dependency of the ribosome-chloroplast associations. This cytoplasmic translation domain aligns localized translation by organellar bacterial-type ribosomes in the chloroplast. This juxtaposition the dual translation systems facilitates the targeting and assembly of the polypeptide products. One-Sentence Summary Translation is localized to a domain of the chloroplast envelope for co-translational protein targeting in Chlamydomonas.

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa , two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa . The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa .

SummaryLignins are cell wall‐located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR‐Cas9‐generated cse1 and cse2 single mutants had a wild‐type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced‐lignin content translated into a fourfold increase in cellulose‐to‐glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p‐coumaroyl, 5‐hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl‐shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

The enzymes that comprise the monolignol biosynthetic pathway have been studied intensively for more than half a century. A major interest has been the role of pathway in the biosynthesis of lignin and the role of lignin in the formation of wood. The pathway has been typically conceived as linear steps that convert phenylalanine into three major monolignols or as a network of enzymes in a metabolic grid. Potential interactions of enzymes have been investigated to test models of metabolic channeling or for higher order interactions. Evidence for enzymatic or physical interactions has been fragmentary and limited to a few enzymes studied in different species. Only recently the entire pathway has been studied comprehensively in any single plant species. Support for interactions comes from new studies of enzyme activity, co-immunoprecipitation, chemical crosslinking, bimolecular fluorescence complementation, yeast 2-hybrid functional screening, and cell type-specific gene expression based on light amplification by stimulated emission of radiation capture microdissection. The most extensive experiments have been done on differentiating xylem of Populus trichocarpa, where genomic, biochemical, chemical, and cellular experiments have been carried out. Interactions affect the rate, direction, and specificity of both 3 and 4-hydroxylation in the monolignol biosynthetic pathway. Three monolignol P450 mono-oxygenases form heterodimeric and heterotetrameric protein complexes that activate specific hydroxylation of cinnamic acid derivatives. Other interactions include regulatory kinetic control of 4-coumarate CoA ligases through subunit specificity and interactions between a cinnamyl alcohol dehydrogenase and a cinnamoyl-CoA reductase. Monolignol enzyme interactions with other pathway proteins have been associated with biotic and abiotic stress response. Evidence challenging or supporting metabolic channeling in this pathway will be discussed.

利用蒸汽爆破法对小茴香秸秆进行处理,研究爆破前后小茴香秸秆常规化学成分和纤维形态的变化及爆破小茴香秸秆对烟草薄片感官品质的影响.结果 表明,在蒸汽爆破压力为1.0 MPa、保压时间3 min的处理条件下,小茴香秸秆纤维出现明显破坏现象,综纤维素和半纤维素含量的降幅分别达到8.1％和15.8％;蒸汽爆破处理有利于小茴香秸秆的打浆及加工生产薄片,可提高产品感官品质.

Intracellular processes can be localized for efficiency or regulation. For example, localized mRNA translation by chloroplastic ribosomes occurs in the biogenesis of PSII, one of the two photosystems of the photosynthetic electron transport chain in the chloroplasts of plants and algae. The biogenesis of PSI and PSII requires the synthesis and assembly of their constituent polypeptide subunits, pigments, and cofactors. Although these biosynthetic pathways are well characterized, less is known about when and where they occur in developing chloroplasts. Here, we used fluorescence microscopy in the unicellular alga Chlamydomonas reinhardtii to reveal spatiotemporal organization in photosystem biogenesis. We focused on translation by chloroplastic ribosomes and chlorophyll biosynthesis in two developmental contexts of active photosystem biogenesis: (1) growth of the mature chloroplast and (2) greening of a nonphotosynthetic chloroplast. The results reveal that a translation zone is the primary location of the biogenesis of PSI and PSII. This discretely localized region within the chloroplast contrasts with the distributions of photosystems throughout this organelle and, therefore, is likely a hub where anabolic pathways converge for photosystem biogenesis.

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO2-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically wasteful oxygenase activity of Rubisco, and mitigates limiting CO2 availability in aquatic environments. Hence, the pyrenoid is functionally analogous to the carboxysomes in cyanobacteria. However, a comprehensive analysis of pyrenoid functions based on its protein composition is lacking. Here we report a proteomic characterization of the pyrenoid in the green alga Chlamydomonas reinhardtii. Pyrenoid-enriched fractions were analyzed by quantitative mass spectrometry. Contaminant proteins were identified by parallel analyses of pyrenoid-deficient mutants. This pyrenoid proteome contains 190 proteins, many of which function in processes that are known or proposed to occur in pyrenoids: e.g. the carbon concentrating mechanism, starch metabolism or RNA metabolism and translation. Using radioisotope pulse labeling experiments, we show that pyrenoid-associated ribosomes could be engaged in the localized synthesis of the large subunit of Rubisco. New pyrenoid functions are supported by proteins in tetrapyrrole and chlorophyll synthesis, carotenoid metabolism or amino acid metabolism. Hence, our results support the long-standing hypothesis that the pyrenoid is a hub for metabolism. The 81 proteins of unknown function reveal candidates for new participants in these processes. Our results provide biochemical evidence of pyrenoid functions and a resource for future research on pyrenoids and their use to enhance agricultural plant productivity. Data are available via ProteomeXchange with identifier PXD004509.

A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.

Magnesium (Mg2+) acts as a cofactor for more than 300 enzymes that are involved in a wide range of fundamental biochemical processes (Knoop et al., 2005Knoop V. Groth-Malonek M. Gebert M. Eifler K. Weyand K. Transport of magnesium and other divalent cations: evolution of the 2-TM-GxN proteins in the MIT superfamily.Mol. Genet. Genomics. 2005; 274: 205-216Crossref PubMed Scopus (110) Google Scholar). In higher plants, Mg2+ has been suggested to play an important role in photosynthesis, including both light reactions and carbon-linked reactions (Portis, 1992Portis A.R. Regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity.Annu. Rev. Plant Biol. 1992; 43: 415-437Crossref Scopus (215) Google Scholar). Despite its important role in photosynthesis, how Mg2+ concentrations are regulated in chloroplasts remains largely unclear. The transmembrane movement of Mg2+ is carried out by Mg2+ transporters (Maguire, 2006Maguire M.E. Magnesium transporters: properties, regulation and structure.Front. Biosci. 2006; 11: 3149-3163Crossref PubMed Scopus (109) Google Scholar). To date, the best characterized plant Mg2+ transporters are the family of CorA, also designated MRS2 or MGT (Schock et al., 2000Schock I. Gregan J. Steinhauser S. Schweyen R. Brennicke A. Knoop V. A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant.Plant J. 2000; 24: 489-501Crossref PubMed Google Scholar, Li et al., 2001Li L. Tutone A.F. Drummond R.S.M. Gardner R.C. Luan S. A novel family of magnesium transport genes in Arabidopsis.Plant Cell. 2001; 13: 2761-2775Crossref PubMed Scopus (242) Google Scholar). In Arabidopsis, all nine MRS2/MGT members have been demonstrated to complement yeast mrs2 mutants (Schock et al., 2000Schock I. Gregan J. Steinhauser S. Schweyen R. Brennicke A. Knoop V. A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant.Plant J. 2000; 24: 489-501Crossref PubMed Google Scholar), but differ in their expression patterns and subcellular localizations (Kobayashi and Tanoi, 2015Kobayashi N.I. Tanoi K. Critical issues in the study of magnesium transport systems and magnesium deficiency symptoms in plants.Int. J. Mol. Sci. 2015; 16: 23076-23093Crossref PubMed Scopus (35) Google Scholar). MGT10/MRS2-11 was observed in the envelope of chloroplasts and expressed mainly in vascular bundles (Drummond et al., 2006Drummond R.S.M. Tutone A. Li Y.C. Gardner R.C. A putative magnesium transporter AtMRS2-11 is localized to the plant chloroplast envelope membrane system.Plant Sci. 2006; 170: 78-89Crossref Scopus (89) Google Scholar, Gebert et al., 2009Gebert M. Meschenmoser K. Svidová S. Weghuber J. Schweyen R. Eifler K. Lenz H. Weyand K. Knoop V. A root-expressed magnesium transporter of the MRS2/MGT gene family in Arabidopsis thaliana allows for growth in low-Mg2+ environments.Plant Cell. 2009; 21: 4018-4030Crossref PubMed Scopus (119) Google Scholar). However, its physiological function remains unknown. Here, we showed that the envelope-localized MGT10 is required for the establishment of Mg2+ concentrations in the stroma of chloroplasts, particularly under high light conditions. In mutant screening for defective chloroplast development, we found a mutant that exhibited the virescence of newly emerged leaves but remained constantly yellow in leaf veins (Figure 1A). In some parts of a leaf, the yellow extended from the vein to mesophyll cells, and green sections looked like small islands (Figure 1A). Genetic analysis revealed that this defective phenotype was controlled by mutations in a single recessive gene, which was mapped finely to an ∼39 kb region, where contains a gene enconding Magnesium Transporter 10 (MGT10), between markers MRN17 and MRN17A (Supplemental Figure 1A). DNA sequencing showed that mgt10 contained a G-to-A transition at +2874 nucleotide from the start codon (ATG) of MGT10, resulting in the formation of a premature stop codon (Supplemental Figure 1A). The map-based cloning result was verified by genetic complementation and also by knocking down expression of MGT10 via artificial miRNA (Figure 1A). RT–PCR analysis showed that mRNA levels of MGT10 were similar among wild-type (WT), mgt10, and two complemented lines, but were reduced significantly in knockdown lines (Supplemental Figure 1B). In addition, transcript levels of other known magnesium transporters/antiporters were not affected by the amiMGT10 transformation, suggesting no off-target effects on those genes (Supplemental Figure 1C). Taken together, our results indicate that the yellow reticulated-leaf vein phenotype results from the mutation in MGT10. The tissue-specific expression patterns of MGT10 were investigated by transforming the reporter gene β-glucuronidase (GUS) driven by the native promoter of MGT10 into the WT plants. Consistent with previous reports (Gebert et al., 2009Gebert M. Meschenmoser K. Svidová S. Weghuber J. Schweyen R. Eifler K. Lenz H. Weyand K. Knoop V. A root-expressed magnesium transporter of the MRS2/MGT gene family in Arabidopsis thaliana allows for growth in low-Mg2+ environments.Plant Cell. 2009; 21: 4018-4030Crossref PubMed Scopus (119) Google Scholar), MGT10 was strongly expressed in the leaf vascular bundle (Supplemental Figure 2). Examination of leaf cross-sections showed that MGT10 was expressed in all cell types, most strongly in the phloem, but not in the xylem vessel (Figure 1B). Such an expression pattern of MGT10 may explain why its mutations lead to a more severe defect of chloroplast development in the vein than in the mesophyll cells. Analysis of subcellular localization via MGT10-GFP fusion protein and western blotting confirmed that MGT10 was targeted to the envelope of chloroplasts (Supplemental Figure 3). Transmission electron microscopy (TEM) analysis showed that thylakoid structure was dramatically altered in the yellow section of mgt10 but was the same as that of WT in the green section of mgt10 (Figure 1C and Supplemental Figure 4). For example, some mgt10 chloroplasts contained grana thylakoids whose length was several-fold longer than that of WT, and some had loosely stacked thylakoids with more membrane layers (Figure 1C). Thus, our results suggest that MGT10 is essential for normal chloroplast development, particularly in the cells around the vascular bundle. The effects of MGT10 mutations on photosynthetic parameters were evaluated by measuring Fv/Fm (the maximum quantum yield of PSII photochemistry), nonphotochemical quenching (NPQ), and millisecond-delayed light emission (ms-DLE, the value of ΔpH across thylakoid membrane). Our results showed that the Fv/Fm value was significantly lower in mgt10 than in WT both in light intensity of 80–100 μmol m−2 s−1 and in high light (600 μmol m−2 s−1) (Figure 1D and Supplemental Figure 5A). Consistently, mgt10 had a slower rate of NPQ induction than WT, but was similar to WT with regard to the relaxation rate (Figure 1E). The significant difference in ms-DLE was only detected in high light between mgt10 and WT (Figure 1F and Supplemental Figure 5B). These results suggest that the ability to protect PSII from light-induced damage is compromised in mgt10. To find out the molecular basis for the hypersensitivity of mgt10 to light, we analyzed photosynthetic complexes by blue native (BN)–PAGE. Under normal light conditions, the major photosynthetic complexes were observed at the same abundance between WT and mgt10, whereas under high light conditions, PSII-related complexes, including PSII supercomplexes and PSII core complexes, were less in mgt10 than in WT (Supplemental Figure 5C). Western blot assay using the specific antibody against a representative subunit of D2 in PSII, PsaD in PSI, Cytf in Cyt b6f, or AtpB in ATP synthase also revealed that the D2 level was mostly reduced in mgt10, which was less than half that of WT under high light conditions (Supplemental Figure 5D). These results indicate that MGT10 plays a role in maintaining PSII activity under high light conditions. Given that MGT10 has been demonstrated to be a magnesium transporter, we proposed that the mgt10 phenotype was caused by Mg2+ deficiency. Unexpectedly, addition of Mg2+ was not able to alleviate the severe yellow cotyledon and retarded growth phenotype of mgt10 seedlings, whereas WT seedlings grew a little bit better with the increase in Mg2+ concentrations up to 10 mM but exhibited growth inhibition or Mg2+ toxicity at concentrations of 20 or 40 mM (Figure 1G). Inductively coupled plasma-mass spectrometry (ICP-MS) analysis indeed showed that mgt10 had higher levels of Mg2+, Ca2+, and Fe than WT (Figure 1H) but had the same level of Cu2+ and Zn2+ as WT (data not shown). Further measurement of Mg2+ concentrations in intact chloroplasts revealed that mgt10 contained 1.75-fold that of the WT Mg2+ level (Figure 1I). To determine whether MGT10 mutations affect Mg2+ transport from roots to shoots, we analyzed Mg2+ content in the xylem sap collected from stems of WT and mgt10 plants supplemented with 0, 10, or 40 mM MgSO4. Our data showed that mgt10 and WT had no statistical difference of Mg2+ content in xylem saps (Figure 1J), implying that MGT10 mutations have no effect on Mg2+ transportation from roots to shoots. Taken together, our data suggest that MGT10 is involved in regulating Mg2+ homeostasis in chloroplasts. The hypersensitivity of mgt10 to light and overaccumulation of Mg2+ in mgt10 chloroplasts prompted us to assess whether low light intensity and sugar could rescue the mgt10 phenotype. As shown in Figure 1K, in low light (40 μmol m−2 s−1), mgt10 cotyledons turned green partially, even on the media without sugar, whereas low light and 1% sugar supplement significantly rescued the mgt10 phenotype. In contrast, when seedlings grew in normal light (100 μmol m−2 s−1), addition or removal of Mg2+ had no obvious effect on the greening of mgt10 cotyledons and leaves. These results suggest that the interaction between light and Mg2+ plays an important role in chloroplast development. In summary, we provided several lines of genetic and physiological evidence demonstrating that MGT10 plays an important role in chloroplast development and photosynthesis. First, mgt10 mutants exhibit yellow reticulated veins and hypersensitivity of PSII activity to high light; second, chloroplasts in the yellow section of mgt10 leaves developed longer grana stacks with more layers of the thylakoid membrane, which may make the damaged PSII move more difficultly to the stroma thylakoids for the repair of D1 protein or reduce photoprotection, and ultimately result in a significant decrease in PSII activity in high light; third, mgt10 chloroplasts accumulated a higher level of Mg2+, indicating that the envelope-localized MGT10 is a Mg2+ transporter from chloroplasts into the cytosol; fourth, we found that low light could significantly rescue the yellow cotyledon phenotype of mgt10. Since we also observed increased content of other ions such as Ca2+ and Fe in mgt10, the possibility cannot be excluded that the observed phenotype of the mutant is also related to the altered levels of these ions. It has been known for a long time that light-induced increase of Mg2+ in the chloroplast stroma is crucial for coordinated regulation of light and dark reactions in photosynthesis (Portis and Heldt, 1976Portis A.R. Heldt H.W. Light-dependent changes of the Mg2+ concentration in the stroma in relation to the Mg2+ dependency of CO2 fixation in intact chloroplasts.Biochim. Biophys. Acta. 1976; 449: 434-446Crossref PubMed Scopus (185) Google Scholar). Our discovery of the physiological role of MGT10 in chloroplast development and photosynthesis lays the basis for elucidating the molecular mechanisms by which Mg2+ levels in the stroma are dynamically regulated in response to various stimuli in the future. This work was supported by grants from the 973 Programs (2015CB910900) and (2015CB150104), and a Chinese National Special Grant for Transgenic Crops (2016ZX08009003-005).

A low-temperature virescent mutant (osv(5a)) was isolated from a 60 Cog-irradiated rice (Oryza sativa) population. At early seedling stage, the mutant exhibits chlorosis phenotype with reduced pigment content at a low temperature (22 degrees C), but it produces green leaves at normal growth temperature (28 degrees C). Chlorophyll accumulation is gradually restored in the mutant at 22 degrees C as it is developing into five-leaf stage. Map-based cloning revealed that OsV5A is a J-like protein with four transmembrane domains. A close homolog, OsV5B, was also identified in the rice genome. Both OsV5A and OsV5B are localized in the chloroplast envelope and thylakoid membranes. We demonstrated that they function as chaperone proteins of protochlorophyllide oxidoreductase (POR), which catalyzes a light-dependent reaction in the chlorophyll biosynthesis pathway. OsV5A and OsV5B interact with two rice PORs (OsPORA and OsPORB) inside chloroplasts and they stabilize OsPORB in vitro under oxidative stress. Differential protein abundances of OsV5A and OsV5B in rice seedlings at different leaf developmental stages were also revealed. OsV5A apparently developed a specialized role for regulating POR abundances at the leaf pre-emergence stage, while the same function is performed by OsV5B during leaf emergence and expansion. Deficiency of OsV5A and OsV5B occurred in pre-emerged and emerging leaves in osv(5a) seedlings at 22 degrees C, leading to reduced POR accumulation and chlorophyll content. Duplication of V5, which is not common among dicots, may have allowed the diversification of their differential roles in regulating chlorophyll biosynthesis in rice and other grass species.