Comparative Microscopic, Transcriptome and IAA Content Analyses Reveal the Stem Growth Variations in Two Cultivars Ilex verticillata.

Ilex verticillata is not only an excellent ornamental tree species for courtyards, but it is also a popular bonsai tree. 'Oosterwijk' and 'Red sprite' are two varieties of Ilex verticillata. The former has a long stem with few branches, while the latter has a short stem. In order to explain the stem growth differences between the two cultivars 'Oosterwijk' and 'Red sprite', determination of the microstructure, transcriptome sequence and IAA content was carried out. The results showed that the xylem thickness, vessel area and vessel number of 'Oosterwijk' were larger than in 'Red sprite'. In addition, our analysis revealed that the differentially expressed genes which were enriched in phenylpropanoid biosynthesis; phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis in the black and tan modules of the two varieties. We found that AST, HCT and bHLH 94 may be key genes in the formation of shoot difference. Moreover, we found that the IAA content and auxin-related DEGs GH3.6, GH3, ATRP5, IAA27, SAUR36-like, GH3.6-like and AIP 10A5-like may play important roles in the formation of shoot differences. In summary, these results indicated that stem growth variations of 'Oosterwijk' and 'Red sprite' were associated with DEGs related to phenylpropanoid biosynthesis, phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis, as well as auxin content and DEGs related to the auxin signaling pathway.

OsMKK6 Regulates Disease Resistance in Rice.

Mitogen-activated protein kinase cascades play important roles in various biological programs in plants, including immune responses, but the underlying mechanisms remain elusive. Here, we identified the lesion mimic mutant rsr25 (rust spots rice 25) and determined that the mutant harbored a loss-of-function allele for OsMKK6 (MITOGEN-ACTIVATED KINASE KINASE 6). rsr25 developed reddish-brown spots on its leaves at the heading stage, as well as on husks. Compared to the wild type, the rsr25 mutant exhibited enhanced resistance to the fungal pathogen Magnaporthe oryzae (M. oryzae) and to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). OsMKK6 interacted with OsMPK4 (MITOGEN-ACTIVATED KINASE 4) in vivo, and OsMKK6 phosphorylated OsMPK4 in vitro. The Osmpk4 mutant is also a lesion mimic mutant, with reddish-brown spots on its leaves and husks. Pathogen-related genes were significantly upregulated in Osmpk4, and this mutant exhibited enhanced resistance to M. oryzae compared to the wild type. Our results indicate that OsMKK6 and OsMPK4 form a cascade that regulates immune responses in rice.

Rice chromatin protein OsHMGB1 is involved in phosphate homeostasis and plant growth by affecting chromatin accessibility.

Although phosphorus is one of the most important essential elements for plant growth and development, the epigenetic regulation of inorganic phosphate (Pi) signaling is poorly understood. In this study, we investigated the biological function and mode of action of the high-mobility-group box 1 protein OsHMGB1 in rice (Oryza sativa), using molecular and genetic approaches. We determined that OsHMGB1 expression is induced by Pi starvation and encodes a nucleus-localized protein. Phenotypic analysis of Oshmgb1 mutant and OsHMGB1 overexpression transgenic plants showed that OsHMGB1 positively regulates Pi homeostasis and plant growth. Transcriptome deep sequencing and chromatin immunoprecipitation followed by sequencing indicated that OsHMGB1 regulates the expression of a series of phosphate starvation-responsive (PSR) genes by binding to their promoters. Furthermore, an assay for transposase-accessible chromatin followed by sequencing revealed that OsHMGB1 is involved in maintaining chromatin accessibility. Indeed, OsHMGB1 occupancy positively correlated with genome-wide chromatin accessibility and gene expression levels. Our results demonstrate that OsHMGB1 is a transcriptional facilitator that regulates the expression of a set of PSR genes to maintain Pi homeostasis in rice by increasing the chromatin accessibility, revealing a key epigenetic mechanism that fine-tune plant acclimation responses to Pi-limited environments.

Effect of Substituents on Molecular Reactivity during Lignin Oxidation by Chlorine Dioxide: A Density Functional Theory Study.

Lignin is a polymer with a complex structure. It is widely present in lignocellulosic biomass, and it has a variety of functional group substituents and linkage forms. Especially during the oxidation reaction, the positioning effect of the different substituents of the benzene ring leads to differences in lignin reactivity. The position of the benzene ring branched chain with respect to methoxy is important. The study of the effect of benzene substituents on the oxidation reaction's activity is still an unfinished task. In this study, density functional theory (DFT) and the m062x/6-311+g (d) basis set were used. Differences in the processes of phenolic oxygen intermediates formed by phenolic lignin structures (with different substituents) with chlorine dioxide during the chlorine dioxide reaction were investigated. Six phenolic lignin model species with different structures were selected. Bond energies, electrostatic potentials, atomic charges, Fukui functions and double descriptors of lignin model substances and reaction energy barriers are compared. The effects of benzene ring branched chains and methoxy on the mechanism of chlorine dioxide oxidation of lignin were revealed systematically. The results showed that the substituents with shorter branched chains and strong electron-absorbing ability were more stable. Lignin is not easily susceptible to the effects of chlorine dioxide. The substituents with longer branched chains have a significant effect on the flow of electron clouds. The results demonstrate that chlorine dioxide can affect the electron arrangement around the molecule, which directly affects the electrophilic activity of the molecule. The electron-absorbing effect of methoxy leads to a low dissociation energy of the phenolic hydroxyl group. Electrophilic reagents are more likely to attack this reaction site. In addition, the stabilizing effect of methoxy on the molecular structure of lignin was also found.

Transcriptome Analysis Reveals the Response Mechanism of Digitaria sanguinalis, Arabidopsis thaliana and Poa annua under 4,8-Dihydroxy-1-tetralone Treatment.

4,8-dihydroxy-l-tetralone (4,8-DHT) is an allelochemical isolated from the outer bark of Carya cathayensis that acts as a plant growth inhibitor. In order to explore the mechanism of 4,8-DHT inhibiting weed activity, we treated three species of Digitaria sanguinalis, Arabidopsis thaliana, and Poa annua with different concentrations of 4,8-DHT and performed phenotype observation and transcriptome sequencing. The results showed that with an increase in 4,8-DHT concentration, the degree of plant damage gradually deepened. Under the same concentration of 4,8-DHT, the damage degree of leaves and roots of Digitaria sanguinalis was the greatest, followed by Arabidopsis thaliana, while Poa annua had the least damage, and the leaves turned slightly yellow. Transcriptome data showed that 24536, 9913, and 1662 differentially expressed genes (DEGs) were identified in Digitaria sanguinalis, Arabidopsis thaliana, and Poa annua, respectively. These DEGs were significantly enriched in photosynthesis, carbon fixation, glutathione metabolism, phenylpropanoid biosynthesis, and oxidative phosphorylation pathways. In addition, DEGs were also enriched in plant hormone signal transduction and the MAPK signal pathway in Arabidopsis thaliana. Further analysis showed that after 4,8-DHT treatment, the transcript levels of photosynthesis PSI- and PSII-related genes, LHCA/B-related genes, Rubisco, and PEPC were significantly decreased in Digitaria sanguinalis and Arabidopsis thaliana. At the same time, the transcription levels of genes related to glutathione metabolism and the phenylpropanoid biosynthesis pathway in Digitaria sanguinalis were also significantly decreased. However, the expression of these genes was upregulated in Arabidopsis thaliana and Poa annua. These indicated that 4,8-DHT affected the growth of the three plants through different physiological pathways, and then played a role in inhibiting plant growth. Simultaneously, the extent to which plants were affected depended on the tested plants and the content of 4,8-DHT. The identification of weed genes that respond to 4,8-DHT has helped us to further understand the inhibition of plant growth by allelochemicals and has provided a scientific basis for the development of allelochemicals as herbicides.

Simultaneous achievement of efficient hemicellulose separation and inhibition of lignin repolymerization using pyruvic acid treatment.

The efficiency of organic acid treatment in the conversion of lignocellulosic biomass fractions has been widely recognized. In this study, a novel green pyruvic acid (PA) treatment is proposed. The higher separation efficiency of eucalyptus hemicellulose was obtained at 4.0% PA and 150 °C. The hemicellulose separation yield was increased from 71.71 to 88.09% compared to glycolic acid (GA) treatment. In addition, the treatment time was significantly reduced from 180 to 40 min. The proportion of cellulose in the solid increased after PA treatment. However, the accompanying separation of lignin was not effectively controlled. Fortunately, a six-membered ring structure was formed on the diol structure of the lignin ß-O-4 side chain. Fewer lignin-condensed structures were observed. High-value lignin rich in phenol hydroxyl groups were obtained. It provides a green path for the simultaneous achievement of efficient hemicellulose separation and inhibition of lignin repolymerization using organic acid treatment.

Integrated transcriptomic analysis identifies coordinated responses to nitrogen and phosphate deficiency in rice.

Nitrogen (N) and phosphorus (P) are two primary components of fertilizers for crop production. Coordinated acquisition and utilization of N and P are crucial for plants to achieve nutrient balance and optimal growth in a changing rhizospheric nutrient environment. However, little is known about how N and P signaling pathways are integrated. We performed transcriptomic analyses and physiological experiments to explore gene expression profiles and physiological homeostasis in the response of rice (Oryza sativa) to N and P deficiency. We revealed that N and P shortage inhibit rice growth and uptake of other nutrients. Gene Ontology (GO) analysis of differentially expressed genes (DEGs) suggested that N and Pi deficiency stimulate specific different physiological reactions and also some same physiological processes in rice. We established the transcriptional regulatory network between N and P signaling pathways based on all DEGs. We determined that the transcript levels of 763 core genes changed under both N or P starvation conditions. Among these core genes, we focused on the transcription factor gene NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) and show that its encoded protein is a positive regulator of P homeostasis and a negative regulator of N acquisition in rice. NIGT1 promoted Pi uptake but inhibited N absorption, induced the expression of Pi responsive genes PT2 and SPX1 and repressed the N responsive genes NLP1 and NRT2.1. These results provide new clues about the mechanisms underlying the interaction between plant N and P starvation responses.

Binding by the Polycomb complex component BMI1 and H2A monoubiquitination shape local and long-range interactions in the Arabidopsis genome.

Three-dimensional (3D) chromatin organization is highly dynamic during development and seems to play a crucial role in regulating gene expression. Self-interacting domains, commonly called topologically associating domains (TADs) or compartment domains (CDs), have been proposed as the basic structural units of chromatin organization. Surprisingly, although these units have been found in several plant species, they escaped detection in Arabidopsis (Arabidopsis thaliana). Here, we show that the Arabidopsis genome is partitioned into contiguous CDs with different epigenetic features, which are required to maintain appropriate intra-CD and long-range interactions. Consistent with this notion, the histone-modifying Polycomb group machinery is involved in 3D chromatin organization. Yet, while it is clear that Polycomb repressive complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) helps establish local and long-range chromatin interactions in plants, the implications of PRC1-mediated histone H2A monoubiquitination on lysine 121 (H2AK121ub) are unclear. We found that PRC1, together with PRC2, maintains intra-CD interactions, but it also hinders the formation of H3K4me3-enriched local chromatin loops when acting independently of PRC2. Moreover, the loss of PRC1 or PRC2 activity differentially affects long-range chromatin interactions, and these 3D changes differentially affect gene expression. Our results suggest that H2AK121ub helps prevent the formation of transposable element/H3K27me1-rich long loops and serves as a docking point for H3K27me3 incorporation.

A novel protein domain is important for photosystem II complex assembly and photoautotrophic growth in angiosperms.

Photosystem II (PSII) is a multi-subunit protein complex of the photosynthetic electron transport chain that is vital to photosynthesis. Although the structure, composition, and function of PSII have been extensively studied, its biogenesis mechanism remains less understood. Thylakoid rhodanese-like (TROL) provides an anchor for leaf-type ferredoxin:NADP+ oxidoreductase. Here, we report the chacterizaton of a second type of TROL protein, TROL2, encoded by seed plant genomes whose function has not previously been reported. We show that TROL2 is a PSII assembly cofactor with essential roles in the establishment of photoautotrophy. TROL2 contains a 45-amino-acid domain, termed the chlorotic lethal seedling (CLS) domain, that is both necessary and sufficient for TROL2 function in PSII assembly and photoautotrophic growth. Phylogenetic analyses suggest that TROL2 may have arisen from ancestral TROL1 via gene duplication before the emergence of seed plants and acquired the CLS domain via evolution of the sequence encoding its N-terminal portion. We further reveal that TROL2 (or CLS) forms an assembly cofactor complex with the intrinsic thylakoid membrane protein LOW PSII ACCUMULATION2 and interacts with small PSII subunits to facilitate PSII complex assembly. Collectively, our study not only shows that TROL2 (CLS) is essential for photoautotrophy in angiosperms but also reveals its mechanistic role in PSII complex assembly, shedding light on the molecular and evolutionary mechanisms of photosynthetic complex assemblyin angiosperms.

Characterizing membrane anchoring of leaf-form ferredoxin-NADP+ oxidoreductase in rice.

Leaf-form ferredoxin-NADP+ oxidoreductases (LFNRs) function in the last step of the photosynthetic electron transport chain, exist as soluble proteins in the chloroplast stroma and are weakly associated with thylakoids or tightly anchored to chloroplast membranes. Arabidopsis thaliana has two LFNRs, and the chloroplast proteins AtTROL and AtTIC62 participate in anchoring AtLFNRs to the thylakoid membrane. By contrast, the membrane anchoring mechanism of rice (Oryza sativa) LFNRs has not been elucidated. Here, we investigated the membrane-anchoring mechanism of LFNRs and its physiological roles in rice. We characterized the rice protein OsTROL1 based on its homology to AtTROL. We determined that OsTROL1 is also a thylakoid membrane anchor and its loss leads to a compensatory increase in OsTIC62. OsLFNR1 attachment through a membrane anchor depends on OsLFNR2, unlike the Arabidopsis counterparts. In addition, OsTIC62 was more highly expressed in the dark than under light conditions, consistent with the increased membrane binding of OsLFNR in the dark. Moreover, we observed reciprocal stabilization between OsLFNRs and their membrane anchors. In addition, unlike in Arabidopsis, the loss of LFNR membrane anchor affects photosynthesis in rice. Overall, our study sheds light on the mechanisms anchoring LFNRs to membranes in rice and highlights differences with Arabidopsis.

Fruit Characteristics of Citrus Trees Grown under Different Soil Cu Levels.

The effects of the increased soil copper (Cu) on fruit quality due to the overuse of Cu agents have been a hot social issue. Seven representative citrus orchards in Guangxi province, China, were investigated to explore the fruit quality characteristics under different soil Cu levels and the relationship between soil-tree Cu and fruit quality. These results showed that pericarp color a value, titratable acid (TA), and vitamin C (Vc) were higher by 90.0, 166.6, and 22.4% in high Cu orchards and by 50.5, 204.2, and 55.3% in excess Cu orchards, compared with optimum Cu orchards. However, the ratio of total soluble solids (TSS)/TA was lower by 68.7% in high Cu orchards and by 61.6% in excess Cu orchards. With the increase of soil Cu concentrations, pericarp color a value and Vc were improved, TA with a trend of rising first then falling, and TSS/TA with a trend of falling first then rising were recorded. As fruit Cu increased, pericarp color a value and TSS reduced and as leaf Cu increased, TSS/TA decreased while Vc was improved. Moreover, a rise in soil Cu enhanced leaf Cu accumulation, and a rise in leaf Cu improved fruit Cu accumulation. Fruit Cu accumulation reduced fruit quality by direct effects, leaf Cu improved fruit quality by direct and indirect effects. Soil Cu affected fruit quality by indirect effects by regulating leaf Cu and fruit Cu. Therefore, reasonable regulation and control of soil Cu concentrations can effectively increase pericarp color, sugar, and acid accumulation in citrus fruit.

Ubiquitin-Specific Protease 2 (OsUBP2) Negatively Regulates Cell Death and Disease Resistance in Rice.

Lesion mimic mutants (LMMs) are great materials for studying programmed cell death and immune mechanisms in plants. Various mechanisms are involved in the phenotypes of different LMMs, but few studies have explored the mechanisms linking deubiquitination and LMMs in rice (Oryza sativa). Here, we identified a rice LMM, rust spots rice (rsr1), resulting from the mutation of a single recessive gene. This LMM has spontaneous reddish-brown spots on its leaves, and displays enhanced resistance to both fungal leaf blast (caused by Magnaporthe oryzae) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). Map-based cloning showed that the mutated gene in rsr1 encodes a Ubiquitin-Specific Protease 2 (OsUBP2). The mutation of OsUBP2 was shown to result in reactive oxygen species (ROS) accumulation, chloroplast structural defects, and programmed cell death, while the overexpression of OsUBP2 weakened rice resistance to leaf blast. OsUBP2 is therefore a negative regulator of immune processes and ROS production. OsUBP2 has deubiquitinating enzyme activity in vitro, and the enzyme active site includes a cysteine at the 234th residue. The ubiquitinated proteomics data of rsr1 and WT provide some possible target protein candidates for OsUBP2.

OsbHLH98 regulates leaf angle in rice through transcriptional repression of OsBUL1.

Leaf angle is an important agronomic trait in cereals that helps determine plant yield by affecting planting density. However, the regulation mechanism of leaf angle remained elusive. Here, we show that OsbHLH98, a rice bHLH transcription factor, negatively regulates leaf angle. osbhlh98 mutant leaves formed a larger leaf angle, whereas transgenic plants overexpressing OsbHLH98 exhibited a slight reduction in leaf angle. We determined that the changes in leaf angle resulted from increased number and size of parenchyma cells on the adaxial side of the lamina joint in osbhlh98 mutants. Experiments using reporter constructs showed that OsbHLH98 is expressed on the adaxial side of lamina joints, consistent with its proposed function in regulating leaf angle. Furthermore, we established by chromatin immunoprecipitation and CUT&RUN that OsBUL1 is a direct downstream target of OsbHLH98. Transactivation assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis indicated that OsbHLH98 represses OsBUL1 transcription. Our results demonstrate that OsbHLH98 negatively regulates leaf angle by counteracting brassinosteroid-induced cell elongation via the repression of OsBUL1 transcription. The characterization of OsbHLH98 and its role in determining leaf angle will lay the foundation to develop the ideal plant architecture for adaptation to high planting density.

H2AK121ub in Arabidopsis associates with a less accessible chromatin state at transcriptional regulation hotspots.

Although it is well established that the Polycomb Group (PcG) complexes maintain gene repression through the incorporation of H2AK121ub and H3K27me3, little is known about the effect of these modifications on chromatin accessibility, which is fundamental to understand PcG function. Here, by integrating chromatin accessibility, histone marks and expression analyses in different Arabidopsis PcG mutants, we show that PcG function regulates chromatin accessibility. We find that H2AK121ub is associated with a less accessible but still permissive chromatin at transcriptional regulation hotspots. Accessibility is further reduced by EMF1 acting in collaboration with PRC2 activity. Consequently, H2AK121ub/H3K27me3 marks are linked to inaccessible although responsive chromatin. In contrast, only-H3K27me3-marked chromatin is less responsive, indicating that H2AK121ub-marked hotspots are required for transcriptional responses. Nevertheless, despite the loss of PcG activities leads to increased chromatin accessibility, this is not necessarily accompanied by transcriptional activation, indicating that accessible chromatin is not always predictive of gene expression.

Preparation and Swelling Behaviors of High-Strength Hemicellulose-g-Polydopamine Composite Hydrogels.

Hemicellulose-based composite hydrogels were successfully prepared by adding polydopamine (PDA) microspheres as reinforcing agents. The effects of PDA microsphere size, dosage, and nitrogen content in hydrogel on the mechanical and rheological properties was studied. The compressive strength of hydrogel was increased from 0.11 to 0.30 MPa. The storage modulus G' was increased from 7.9 to 22.0 KPa. The gaps in the hemicellulose network are filled with PDA microspheres. There is also chemical cross-linking between them. These gaps increased the density of the hydrogel network structure. It also has good water retention and pH sensitivity. The maximum cumulative release rate of methylene blue was 62.82%. The results showed that the release behavior of hydrogel was pH-responsive, which was beneficial to realizing targeted and controlling drug release.

OsbHLH6 interacts with OsSPX4 and regulates the phosphate starvation response in rice.

Low soil phosphorus (P) availability is a major limitation for crop production. The molecular mechanisms underlying plant responses and adaptation to phosphate (Pi) deficiency are unclear. OsbHLH6 (hereafter bHLH6), an uncharacterized rice (Oryza sativa) Pi starvation response gene encoding a basic helix-loop-helix protein, was identified by yeast two-hybrid screening using the phosphate response repressor OsSPX4 (hereafter SPX4) as bait. bHLH6 is expressed in shoots and roots, and its expression is significantly induced in shoots by Pi deficiency. bHLH6 overexpression lines showed Pi accumulation and enhanced Pi starvation responses, including upregulation of Pi starvation-induced genes and longer root hairs. A bhlh6 mutant showed no significant phenotype variation at the seedling stage. A pull-down assay indicated that bHLH6 had higher binding affinity with SPX4 compared to OsPHR2; therefore, bHLH6 competitively inhibited the interaction of SPX4 and OsPHR2. SPX4 overexpression rescued the Pi accumulation caused by bHLH6 overexpression under high- and low-P conditions. Moreover, overexpression of bHLH6 in an spx4 background did not affect the Pi content of spx4 under high- and low-P conditions. The bhlh6 spx4 double mutant showed lower shoot Pi concentrations and transcript levels of OsPT3 and OsPT10 compared with the spx4 mutant under high-P conditions. RNA sequencing results indicated that bHLH6 overexpression and spx4 mutant lines share many differentially expressed Pi-responsive genes. Therefore, bHLH6 is an important regulator for Pi signaling and homeostasis which antagonizes SPX4. This knowledge helps elucidate the molecular regulation of plant adaptation to Pi deficiency and will promote efforts toward the creation of low Pi-tolerant crops.

CRD1, an Xpo1 domain protein, regulates miRNA accumulation and crown root development in rice.

Crown root (CR) is the main component of the fibrous root system in cereal crops, but the molecular mechanism underlying CR development is still unclear. Here, we isolated the crown root defect 1 (crd1) mutant from ethyl methane sulfonate-mutated mutant library, which significantly inhibited CR development. The CRD1 was identified through genome resequencing and complementation analysis, which encodes an Xpo1 domain protein: the rice ortholog of Arabidopsis HASTY (HST) and human exportin-5 (XPO5). CRD1 is ubiquitously expressed, with the highest expression levels in the CR primordium at the stem base. CRD1 is a nucleocytoplasmic protein. The crd1 mutant contains significantly reduced miRNA levels in the cytoplasm and nucleus, suggesting that CRD1 is essential for maintaining normal miRNA levels in plant cells. The altered CR phenotype of crd1 was simulated by target mimicry of miR156, suggesting that this defect is due to the disruption of miR156 regulatory pathways. Our analysis of CRD1, the HST ortholog identified in monocots, expands our understanding of the molecular mechanisms underlying miRNA level and CR development.

LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice.

Root system architecture is very important for plant growth and crop yield. It is essential for nutrient and water uptake, anchoring, and mechanical support. Root growth angle (RGA) is a vital constituent of root system architecture and is used as a parameter for variety evaluation in plant breeding. However, little is known about the underlying molecular mechanisms that determine root growth angle in rice (Oryza sativa). In this study, a rice mutant large root angle1 (lra1) was isolated and shown to exhibit a large RGA and reduced sensitivity to gravity. Genome resequencing and complementation assays identified OsPIN2 as the gene responsible for the mutant phenotypes. OsPIN2 was mainly expressed in roots and the base of shoots, and showed polar localization in the plasma membrane of root epidermal and cortex cells. OsPIN2 was shown to play an important role in mediating root gravitropic responses in rice and was essential for plants to produce normal RGAs. Taken together, our findings suggest that OsPIN2 plays an important role in root gravitropic responses and determining the root system architecture in rice by affecting polar auxin transport in the root tip.

LIGHT-INDUCED RICE1 Regulates Light-Dependent Attachment of LEAF-TYPE FERREDOXIN-NADP+ OXIDOREDUCTASE to the Thylakoid Membrane in Rice and Arabidopsis.

LIR1 (LIGHT-INDUCED RICE1) encodes a 13-kD, chloroplast-targeted protein containing two nearly identical motifs of unknown function. LIR1 is present in the genomes of vascular plants, mosses, liverworts, and algae, but not in cyanobacteria. Using coimmunoprecipitation assays, pull-down assays, and yeast two-hybrid analyses, we showed that LIR1 interacts with LEAF-TYPE FERREDOXIN-NADP(+) OXIDOREDUCTASE (LFNR), an essential chloroplast enzyme functioning in the last step of photosynthetic linear electron transfer. LIR1 and LFNR formed high molecular weight thylakoid protein complexes with the TIC62 and TROL proteins, previously shown to anchor LFNR to the membrane. We further showed that LIR1 increases the affinity of LFNRs for TIC62 and that the rapid light-triggered degradation of the LIR1 coincides with the release of the LFNR from the thylakoid membrane. Loss of LIR1 resulted in a marked decrease in the accumulation of LFNR-containing thylakoid protein complexes without a concomitant decrease in total LFNR content. In rice (Oryza sativa), photosynthetic capacity of lir1 plants was slightly impaired, whereas no such effect was observed in Arabidopsis thaliana knockout mutants. The consequences of LIR1 deficiency in different species are discussed.

Integrative Comparison of the Role of the PHOSPHATE RESPONSE1 Subfamily in Phosphate Signaling and Homeostasis in Rice.

Phosphorus (P), an essential macronutrient for all living cells, is indispensable for agricultural production. Although Arabidopsis (Arabidopsis thaliana) PHOSPHATE RESPONSE1 (PHR1) and its orthologs in other species have been shown to function in transcriptional regulation of phosphate (Pi) signaling and Pi homeostasis, an integrative comparison of PHR1-related proteins in rice (Oryza sativa) has not previously been reported. Here, we identified functional redundancy among three PHR1 orthologs in rice (OsPHR1, OsPHR2, and OsPHR3) using phylogenetic and mutation analysis. OsPHR3 in conjunction with OsPHR1 and OsPHR2 function in transcriptional activation of most Pi starvation-induced genes. Loss-of-function mutations in any one of these transcription factors (TFs) impaired root hair growth (primarily root hair elongation). However, these three TFs showed differences in DNA binding affinities and messenger RNA expression patterns in different tissues and growth stages, and transcriptomic analysis revealed differential effects on Pi starvation-induced gene expression of single mutants of the three TFs, indicating some degree of functional diversification. Overexpression of genes encoding any of these TFs resulted in partial constitutive activation of Pi starvation response and led to Pi accumulation in the shoot. Furthermore, unlike OsPHR2-overexpressing lines, which exhibited growth retardation under normal or Pi-deficient conditions, OsPHR3-overexpressing plants exhibited significant tolerance to low-Pi stress but normal growth rates under normal Pi conditions, suggesting that OsPHR3 would be useful for molecular breeding to improve Pi uptake/use efficiency under Pi-deficient conditions. We propose that OsPHR1, OsPHR2, and OsPHR3 form a network and play diverse roles in regulating Pi signaling and homeostasis in rice.