MATE transporter GFD1 cooperates with sugar transporters, mediates carbohydrate partitioning and controls grain-filling duration, grain size and number in rice.

More than half of the world's food is provided by cereals, as humans obtain >60% of daily calories from grains. Producing more carbohydrates is always the final target of crop cultivation. The carbohydrate partitioning pathway directly affects grain yield, but the molecular mechanisms and biological functions are poorly understood, including rice (Oryza sativa L.), one of the most important food sources. Here, we reported a prolonged grain filling duration mutant 1 (gfd1), exhibiting a long grain-filling duration, less grain number per panicle and bigger grain size without changing grain weight. Map-based cloning and molecular biological analyses revealed that GFD1 encoded a MATE transporter and expressed high in vascular tissues of the stem, spikelet hulls and rachilla, but low in the leaf, controlling carbohydrate partitioning in the stem and grain but not in the leaf. GFD1 protein was partially localized on the plasma membrane and in the Golgi apparatus, and was finally verified to interact with two sugar transporters, OsSWEET4 and OsSUT2. Genetic analyses showed that GFD1 might control grain-filling duration through OsSWEET4, adjust grain size with OsSUT2 and synergistically modulate grain number per panicle with both OsSUT2 and OsSWEET4. Together, our work proved that the three transporters, which are all initially classified in the major facilitator superfamily family, could control starch storage in both the primary sink (grain) and temporary sink (stem), and affect carbohydrate partitioning in the whole plant through physical interaction, giving a new vision of sugar transporter interactome and providing a tool for rice yield improvement.

Ef-cd locus shortens rice maturity duration without yield penalty.

The contradiction between "high yielding" and "early maturing" hampers further improvement of annual rice yield. Here we report the positional cloning of a major maturity duration regulatory gene, Early flowering-completely dominant (Ef-cd), and demonstrate that natural variation in Ef-cd could be used to overcome the above contradictory. The Ef-cd locus gives rise to a long noncoding RNA (lncRNA) antisense transcript overlapping the OsSOC1 gene. Ef-cd lncRNA expression positively correlates with the expression of OsSOC1 and H3K36me3 deposition. Field test comparisons of early maturing Ef-cd near-isogenic lines with their wild types as well as of the derivative early maturing hybrids with their wild-type hybrids conducted under different latitudes determined that the early maturing Ef-cd allele shortens maturity duration (ranging from 7 to 20 d) without a concomitant yield penalty. Ef-cd facilitates nitrogen utilization and also improves the photosynthesis rate. Analysis of 1,439 elite hybrid rice varieties revealed that the 16 homozygotes and 299 heterozygotes possessing Ef-cd matured significantly earlier. Therefore, Ef-cd could be a vital contributor of elite early maturing hybrid varieties in balancing grain yield with maturity duration.

DRB2 Modulates Leaf Rolling by Regulating Accumulation of MicroRNAs Related to Leaf Development in Rice.

As an important agronomic trait in rice (Oryza sativa), moderate leaf rolling helps to maintain the erectness of leaves and minimize shadowing between leaves, leading to improved photosynthetic efficiency and grain yield. However, the molecular mechanisms underlying rice leaf rolling still need to be elucidated. Here, we isolated a rice mutant, rl89, showing adaxially rolled leaf phenotype due to decreased number and size of bulliform cells. We confirmed that the rl89 phenotypes were caused by a single nucleotide substitution in OsDRB2 (LOC_Os10g33970) gene encoding DOUBLE-STRANDED RNA-BINDING2. This gene was constitutively expressed, and its encoded protein was localized to both nucleus and cytoplasm. Yeast two-hybrid assay showed that OsDRB2 could interact with DICER-LIKE1 (DCL1) and OsDRB1-2 respectively. qRT-PCR analysis of 29 related genes suggested that defects of the OsDRB2-miR166-OsHBs pathway could play an important role in formation of the rolled leaf phenotype of rl89, in which OsDRB2 mutation reduced miR166 accumulation, resulting in elevated expressions of the class III homeodomain-leucine zipper genes (such as OsHB1, 3 and 5) involved in leaf polarity and/or morphology development. Moreover, OsDRB2 mutation also reduced accumulation of miR160, miR319, miR390, and miR396, which could cause the abnormal leaf development in rl89 by regulating expressions of their target genes related to leaf development.

Mutation of Protoporphyrinogen IX Oxidase Gene Causes Spotted and Rolled Leaf and Its Overexpression Generates Herbicide Resistance in Rice.

Protoporphyrinogen IX (Protogen IX) oxidase (PPO) catalyzes the oxidation of Protogen IX to Proto IX. PPO is also the target site for diphenyl ether-type herbicides. In plants, there are two PPO encoding genes, PPO1 and PPO2. To date, no PPO gene or mutant has been characterized in monocotyledonous plants. In this study, we isolated a spotted and rolled leaf (sprl1) mutant in rice (Oryza sativa). The spotted leaf phenotype was sensitive to high light intensity and low temperature, but the rolled leaf phenotype was insensitive. We confirmed that the sprl1 phenotypes were caused by a single nucleotide substitution in the OsPPO1 (LOC_Os01g18320) gene. This gene is constitutively expressed, and its encoded product is localized to the chloroplast. The sprl1 mutant accumulated excess Proto(gen) IX and reactive oxygen species (ROS), resulting in necrotic lesions. The expressions of 26 genes associated with tetrapyrrole biosynthesis, photosynthesis, ROS accumulation, and rolled leaf were significantly altered in sprl1, demonstrating that these expression changes were coincident with the mutant phenotypes. Importantly, OsPPO1-overexpression transgenic plants were resistant to the herbicides oxyfluorfen and acifluorfen under field conditions, while having no distinct influence on plant growth and grain yield. These finding indicate that the OsPPO1 gene has the potential to engineer herbicide resistance in rice.

Crosstalk between the Circadian Clock and Histone Methylation.

The circadian clock and histone modifications could form a feedback loop in Arabidopsis; whether a similar regulatory mechanism exists in rice is still unknown. Previously, we reported that SDG724 and OsLHY are two rice heading date regulators in rice. SDG724 encodes a histone H3K36 methyltransferase, and OsLHY is a vital circadian rhythm transcription factor. Both could be involved in transcription regulatory mechanisms and could affect gene expression in various pathways. To explore the crosstalk between the circadian clock and histone methylation in rice, we studied the relationship between OsLHY and SDG724 via the transcriptome analysis of their single and double mutants, oslhy, sdg724, and oslhysdg724. Screening of overlapped DEGs and KEGG pathways between OsLHY and SDG724 revealed that they could control many overlapped pathways indirectly. Furthermore, we identified three candidate targets (OsGI, OsCCT38, and OsPRR95) of OsLHY and one candidate target (OsCRY1a) of SDG724 in the clock pathway. Our results showed a regulatory relationship between OsLHY and SDG724, which paved the way for revealing the interaction between the circadian clock and histone H3K36 methylation.

Dual function of clock component OsLHY sets critical day length for photoperiodic flowering in rice.

Circadian clock, an endogenous time-setting mechanism, allows plants to adapt to unstable photoperiod conditions and induces flowering with proper timing. In Arabidopsis, the central clock oscillator was formed by a series of interlocked transcriptional feedback loops, but little is known in rice so far. By MutMap technique, we identified the candidate gene OsLHY from a later flowering mutant lem1 and further confirmed it through genetic complementation, RNA interference knockdown, and CRISPR/Cas9-knockout. Global transcriptome profiling and expression analyses revealed that OsLHY might be a vital circadian rhythm component. Interestingly, oslhy flowered later under ≥12 h day length but headed earlier under ≤11 h day length. qRT-PCR results exhibited that OsLHY might function through OsGI-Hd1 pathway. Subsequent one-hybrid assays in yeast, DNA affinity purification qPCR, and electrophoretic mobility shift assays confirmed OsLHY could directly bind to the CBS element in OsGI promoter. Moreover, the critical day length (CDL) for function reversal of OsLHY in oslhy (11-12 h) was prolonged in the double mutant oslhy osgi (about 13.5 h), indicating that the CDL set by OsLHY was OsGI dependent. Additionally, the dual function of OsLHY entirely relied on Hd1, as the double mutant oslhy hd1 showed the same heading date with hd1 under about 11.5, 13.5, and 14 h day lengths. Together, OsLHY could fine-tune the CDL by directly regulating OsGI, and Hd1 acts as the final effector of CDL downstream of OsLHY. Our study illustrates a new regulatory mechanism between the circadian clock and photoperiodic flowering.

A single nucleotide substitution at the 3'-end of SBPase gene involved in Calvin cycle severely affects plant growth and grain yield in rice.

BACKGROUND: Calvin cycle plays a crucial role in carbon fixation which provides the precursors of organic macromolecules for plant growth and development. Currently, no gene involved in Calvin cycle has been identified in monocotyledonous plants through mutant or/and map-based cloning approach. RESULTS: Here, we isolated a low-tillering mutant, c6635, in rice (Oryza sativa). The mutant displayed light green leaves and intensely declined pigment contents and photosynthetic capacity at early growth stage. Moreover, its individual plant showed a much smaller size, and most individuals produced only two tillers. At mature stage, its productive panicles, grain number and seed setting rate were significantly decreased, which lead to a sharp reduction of the grain yield. We confirmed that a single nucleotide mutation in LOC_Os04g16680 gene encoding sedoheptulose 1,7-bisphosphatase (SBPase) involved in Calvin cycle was responsible for the mutant phenotype of c6635 through map-based cloning, MutMap analysis and complementation experiments. Sequence analysis suggested that the point mutation caused an amino acid change from Gly-364 to Asp at the C-terminal of SBPase. In addition, OsSBPase gene was mainly expressed in leaf, and the encoded protein was located in chloroplast. The mutation of OsSBPase could significantly affect expression levels of some key genes involved in Calvin cycle. CONCLUSIONS: We successfully identified a SBPase gene in monocotyledonous plants. Meanwhile, we demonstrated that a single nucleotide substitution at the 3'-end of this gene severely affects plant growth and grain yield, implying that the Gly-364 at the C-terminal of SBPase could play an important role in SBPase function in rice.

LPS1, Encoding Iron-Sulfur Subunit SDH2-1 of Succinate Dehydrogenase, Affects Leaf Senescence and Grain Yield in Rice.

The iron-sulfur subunit (SDH2) of succinate dehydrogenase plays a key role in electron transport in plant mitochondria. However, it is yet unknown whether SDH2 genes are involved in leaf senescence and yield formation. In this study, we isolated a late premature senescence mutant, lps1, in rice (Oryza sativa). The mutant leaves exhibited brown spots at late tillering stage and wilted at the late grain-filling stage and mature stage. In its premature senescence leaves, photosynthetic pigment contents and net photosynthetic rate were reduced; chloroplasts and mitochondria were degraded. Meanwhile, lps1 displayed small panicles, low seed-setting rate and dramatically reduced grain yield. Gene cloning and complementation analysis suggested that the causal gene for the mutant phenotype was OsSDH2-1 (LOC_Os08g02640), in which single nucleotide mutation resulted in an amino acid substitution in the encoded protein. OsSDH2-1 gene was expressed in all organs tested, with higher expression in leaves, root tips, ovary and anthers. OsSDH2-1 protein was targeted to mitochondria. Furthermore, reactive oxygen species (ROS), mainly H2O2, was excessively accumulated in leaves and young panicles of lps1, which could cause premature leaf senescence and affect panicle development and pollen function. Taken together, OsSDH2-1 plays a crucial role in leaf senescence and yield formation in rice.

A lil3 chlp double mutant with exclusive accumulation of geranylgeranyl chlorophyll displays a lethal phenotype in rice.

BACKGROUND: Phytyl residues are the common side chains of chlorophyll (Chl) and tocopherols. Geranylgeranyl reductase (GGR), which is encoded by CHLP gene, is responsible for phytyl biosynthesis. The light-harvesting like protein LIL3 was suggested to be required for stability of GGR and protochlorophyllide oxidoreductase in Arabidopsis. RESULTS: In this study, we isolated a yellow-green leaf mutant, 637ys, in rice (Oryza sativa). The mutant accumulated majority of Chls with unsaturated geranylgeraniol side chains and displayed a yellow-green leaf phenotype through the whole growth period. The development of chloroplasts was suppressed, and the major agronomic traits, especially No. of productive panicles per plant and of spikelets per panicle, dramatically decreased in 637ys. Besides, the mutant exhibited to be sensitive to light intensity and deficiency of tocopherols without obvious alteration in tocotrienols in leaves and grains. Map-based cloning and complementation experiment demonstrated that a point mutation on the OsLIL3 gene accounted for the mutant phenotype of 637ys. OsLIL3 is mainly expressed in green tissues, and its encoded protein is targeted to the chloroplast. Furthermore, the 637ys 502ys (lil3 chlp) double mutant exclusively accumulated geranylgeranyl Chl and exhibited lethality at the three-leaf stage. CONCLUSIONS: We identified the OsLIL3 gene through a map-based cloning approach. Meanwhile, we demonstrated that OsLIL3 is of extreme importance to the function of OsGGR, and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.

A single nucleotide mutation of IspF gene involved in the MEP pathway for isoprenoid biosynthesis causes yellow-green leaf phenotype in rice.

KEY MESSAGE: We identified IspF gene through yellow-green leaf mutant 505ys in rice. OsIspF was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. On expression levels of genes in this mutant, OsIspF itself and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase were all up-regulated, however, among eight genes associated with photosynthesis, only psaA, psaN and psbA genes for three reaction center subunits of photosystem obviously changed. Isoprenoids are the most abundant natural compounds in all organisms, which originate from the basic five-carbon units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, IPP and DMAPP are synthesized through two independent pathways, the mevalonic acid pathway in cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. The MEP pathway comprises seven enzymatic steps, in which IspF is the fifth enzyme. So far, no IspF gene has been identified in monocotyledonous plants. In this study, we isolated a leaf-color mutant, 505ys, in rice (Oryza sativa). The mutant displayed yellow-green leaf phenotype, reduced level of photosynthetic pigments, and arrested development of chloroplasts. By map-based cloning of this mutant, we identified OsIspF gene (LOC_Os02g45660) showing significant similarity to IspF gene of Arabidopsis, in which a missense mutation occurred in the mutant, resulting in an amino acid change in the encoded protein. OsIspF gene was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. Further, the mutant phenotype of 505ys was complemented by transformation with the wild-type OsIspF gene. Therefore, we successfully identified an IspF gene in monocotyledonous plants. In addition, real-time quantitative RT-PCR implied that a positive regulation could exist between the OsIspF gene and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase. At the same time, it also implied that the individual genes involved in the MEP pathway might differentially regulated expression levels of the genes associated with photosynthesis.

A Single Nucleotide Mutation of the IspE Gene Participating in the MEP Pathway for Isoprenoid Biosynthesis Causes a Green-Revertible Yellow Leaf Phenotype in Rice.

Plant isoprenoids are dependent on two independent pathways, the cytosolic mevalonate (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. IspE is one of seven known enzymes in the MEP pathway. Currently, no IspE gene has been identified in rice. In addition, no virescent mutants have been reported to result from a gene mutation affecting the MEP pathway. In this study, we isolated a green-revertible yellow leaf mutant gry340 in rice. The mutant exhibited a reduced level of photosynthetic pigments, and an arrested development of chloroplasts and mitochondria in its yellow leaves. Map-based cloning revealed a missense mutation in OsIspE (LOC_Os01g58790) in gry340 mutant plants. OsIspE is constitutively expressed in all tissues, and its encoded protein is targeted to the chloroplast. Further, the mutant phenotype of gry340 was rescued by introduction of the wild-type gene. Therefore, we have successfully identified an IspE gene in monocotyledons via map-based cloning, and confirmed that the green-revertible yellow leaf phenotype of gry340 does result from a single nucleotide mutation in the IspE gene. In addition, the ispE ispF double mutant displayed an etiolation lethal phenotype, indicating that the isoprenoid precursors from the cytosol cannot efficiently compensate for the deficiency of the MEP pathway in rice chloroplasts. Furthermore, real-time quantitative reverse transcription-PCR suggested that this functional defect in OsIspE affected the expression of not only other MEP pathway genes but also that of MVA pathway genes, photosynthetic genes and mitochondrial genes.

Molecular characterization and functional analysis of the OsPsbR gene family in rice.

Low temperature may exert a negative impact on agronomical productivity. PsbR was known as the 10 kDa Photosystem II polypeptide. Although plant PsbR is thought to play important roles in photosynthesis, little is known about the contribution of plant PsbR to abiotic stress resistance. The expression patterns of three OsPsbR gene family members, OsPsbR1, OsPsbR2, and OsPsbR3, were characterized in rice 'Nipponbare'. Under normal condition, OsPsbR1 and OsPsbR3 showed tissue-specific expression, while the expression of OsPsbR2 could not be detected in all tested tissues. OsPsbR1 was upregulated in response to cold stress, and downregulated under drought, salt, or heat conditions. The upregulation of OsPsbR3 was observed under the treatment of ABA, and its downregulation was detected under drought or heat conditions. Upregulation of OsPsbR1 in rice resulted in significantly increased resistance to cold, but did not affect the yield of rice. Furthermore, after 8 h cold-stress treatment, the expression levels of three cold stress-induced marker genes were significantly higher in the overexpression lines L11 and L19 in comparison with the wild type. All these results suggest that OsPsbR1 may play key roles in photosynthesis and cold stress response and thus has the potential to improve cold stress tolerance of crops.

Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae.

MicroRNAs (miRNAs) are indispensable regulators for development and defense in eukaryotes. However, the miRNA species have not been explored for rice (Oryza sativa) immunity against the blast fungus Magnaporthe oryzae, the most devastating fungal pathogen in rice production worldwide. Here, by deep sequencing small RNA libraries from susceptible and resistant lines in normal conditions and upon M. oryzae infection, we identified a group of known rice miRNAs that were differentially expressed upon M. oryzae infection. They were further classified into three classes based on their expression patterns in the susceptible japonica line Lijiangxin Tuan Hegu and in the resistant line International Rice Blast Line Pyricularia-Kanto51-m-Tsuyuake that contains a single resistance gene locus, Pyricularia-Kanto 51-m (Pikm), within the Lijiangxin Tuan Hegu background. RNA-blot assay of nine of them confirmed sequencing results. Real-time reverse transcription-polymerase chain reaction assay showed that the expression of some target genes was negatively correlated with the expression of miRNAs. Moreover, transgenic rice plants overexpressing miR160a and miR398b displayed enhanced resistance to M. oryzae, as demonstrated by decreased fungal growth, increased hydrogen peroxide accumulation at the infection site, and up-regulated expression of defense-related genes. Taken together, our data indicate that miRNAs are involved in rice immunity against M. oryzae and that overexpression of miR160a or miR398b can enhance rice resistance to the disease.

GRY79 encoding a putative metallo-ß-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice.

KEY MESSAGE: The green - revertible yellow79 mutant resulting from a single-base mutation suggested that the GRY79 gene encoding a putative metallo-ß-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice. Functional studies of metallo-ß-lactamases and trihelix transcription factors in higher plants remain very sparse. In this study, we isolated the green-revertible yellow79 (gry79) mutant in rice. The mutant developed yellow-green leaves before the three-leaf stage but recovered to normal green at the sixth-leaf stage. Meanwhile, the mutant exhibited reduced level of chlorophylls and arrested development of chloroplasts in the yellow leaves. Genetic analysis suggested that the mutant phenotype was controlled by a single recessive nuclear gene on rice chromosome 2. Map-based cloning revealed that the candidate gene was Os02g33610 encoding a putative metallo-ß-lactamase-trihelix chimera. In the gry79 mutant, a single-base mutation occurred in coding region of the gene, resulting in an amino acid change in the encoded protein. Furthermore, the mutant phenotype was rescued by transformation with the wild-type gene. Therefore, we have confirmed that the gry79 mutant phenotype resulted from a single-base mutation in GRY79 (Os02g33610) gene, suggesting that the gene encoding a putative metallo-ß-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice. In addition, we considered that the gry79 mutant gene could be applicable as a leaf-color marker gene for efficient identification and elimination of false hybrids in commercial hybrid rice production.

One divinyl reductase reduces the 8-vinyl groups in various intermediates of chlorophyll biosynthesis in a given higher plant species, but the isozyme differs between species.

Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous.

Fenchone Ameliorates Constipation-Predominant Irritable Bowel Syndrome via Modulation of SCF/c-Kit Pathway and Gut Microbiota.

In this study we sought to elucidate the therapeutic effects of fenchone on constipation-predominant irritable bowel syndrome (IBS-C) and the underlying mechanisms. An IBS-C model was established in rats by administration of ice water by gavage for 14 days. Fenchone increased the reduced body weight, number of fecal pellets, fecal moisture, and intestinal transit rate, and decreased the enhanced visceral hypersensitivity in the rat model of IBS-C. In addition, fenchone increased the serum content of excitatory neurotransmitters and decreased the serum content of inhibitory neurotransmitters in the IBS-C rat model. Meanwhile, western blot and immunofluorescence experiments indicated that fenchone increased the expressions of SCF and c-Kit. Furthermore, compared with the IBS-C model group, fenchone increased the relative abundance of Lactobacillus, Blautia, Allobaculum, Subdoligranulum, and Ruminococcaceae_UCG-008, and reduced the relative abundance of Bacteroides, Enterococcus, Alistipes, and Escherichia-Shigella on the genus level. Overall, fenchone ameliorates IBS-C via modulation of the SCF/c-Kit pathway and gut microbiota, and could therefore serve as a novel drug candidate against IBS-C.

The impact of protein delivery on short-term and long-term clinical outcomes in critically ill patients: Protocol for a multicenter, prospective, observational study (The ACTION study).

BACKGROUND AND AIMS: Nutrition therapy is a vital part of the management of critically ill patients. Efforts have been made to optimize nutrition therapy in the ICU setting, and it is argued that protein might be the most important substrate to deliver during critical illness. However, the impact of protein delivery on patient-centered outcomes, including short-term and long-term outcomes, is controversial. Moreover, previous studies showed that compliance with the guidelines is poor in practice, and the amounts of protein intake vary significantly among different hospitals. The objective of this study is to describe the current practice of protein delivery for critically ill patients and to investigate the association between different protein delivery amounts and approaches during ICU admission and multiple patient-centered outcomes (short-term and long-term). METHODS: This is a multicenter, prospective, observational study conducted in 70 hospitals, aiming to recruit more than 1800 newly admitted critically ill patients who are expected to stay in ICU for at least 48 h. Data, including the baseline characteristics, illness severity scores, requirements of organ support therapy, and daily nutritional therapy, will be recorded until day 28 after enrollment unless discharge from the ICU or death occurs first. The key long-term clinical outcomes, like readmission post the index discharge and health-related quality of life, will be collected via telephone contact on Day 90 and Day 180 after recruitment. Quality of life will be assessed by the EuroQol five dimensions five-level questionnaire (EQ5D5L) visual analogue scale score. Apart from descriptive data, multivariate analyses adjusted for potential confounders will be applied to assess the association between protein intake during ICU stay and short-term and long-term clinical outcomes. ETHICS AND TRIAL REGISTRATION: This study was reviewed and approved by the ethics committee of Jinling Hospital (2021NZKY-027-01) and the participating sites. The study was registered at the Chinese Clinical Trials Registry (ChiCTR2200067016) before enrollment.

Research progress and application strategies of sugar transport mechanisms in rice.

In plants, carbohydrates are central products of photosynthesis. Rice is a staple that contributes to the daily calorie intake for over half of the world's population. Hence, the primary objective of rice cultivation is to maximize carbohydrate production. The "source-sink" theory is proposed as a valuable principle for guiding crop breeding. However, the "flow" research lag, especially in sugar transport, has hindered high-yield rice breeding progress. This review concentrates on the genetic and molecular foundations of sugar transport and its regulation, enhancing the fundamental understanding of sugar transport processes in plants. We illustrate that the apoplastic pathway is predominant over the symplastic pathway during phloem loading in rice. Sugar transport proteins, such as SUTs and SWEETs, are essential carriers for sugar transportation in the apoplastic pathway. Additionally, we have summarized a regulatory pathway for sugar transport genes in rice, highlighting the roles of transcription factors (OsDOF11, OsNF-YB1, OsNF-YC12, OsbZIP72, Nhd1), OsRRM (RNA Recognition Motif containing protein), and GFD1 (Grain Filling Duration 1). Recognizing that the research shortfall in this area stems from a lack of advanced research methods, we discuss cutting-edge analytical techniques such as Mass Spectrometry Imaging and single-cell RNA sequencing, which could provide profound insights into the dynamics of sugar distribution and the associated regulatory mechanisms. In summary, this comprehensive review serves as a valuable guide, directing researchers toward a deep understanding and future study of the intricate mechanisms governing sugar transport.

OsPOP5, a prolyl oligopeptidase family gene from rice confers abiotic stress tolerance in Escherichia coli.

The prolyl oligopeptidase family, which is a group of serine peptidases, can hydrolyze peptides smaller than 30 residues. The prolyl oligopeptidase family in plants includes four members, which are prolyl oligopeptidase (POP, EC3.4.21.26), dipeptidyl peptidase IV (DPPIV, EC3.4.14.5), oligopeptidase B (OPB, EC3.4.21.83), and acylaminoacyl peptidase (ACPH, EC3.4.19.1). POP is found in human and rat, and plays important roles in multiple biological processes, such as protein secretion, maturation and degradation of peptide hormones, and neuropathies, signal transduction and memory and learning. However, the function of POP is unclear in plants. In order to study POP function in plants, we cloned the cDNA of the OsPOP5 gene from rice by nested-PCR. Sequence analysis showed that the cDNA encodes a protein of 596 amino acid residues with Mw ≈ 67.29 kD. In order to analyze the protein function under different abiotic stresses, OsPOP5 was expressed in Escherichia coli. OsPOP5 protein enhanced the tolerance of E. coli to high salinity, high temperature and simulated drought. The results indicate that OsPOP5 is a stress-related gene in rice and it may play an important role in plant tolerance to abiotic stress.

Co3O4/CoFe2O4 Hollow Nanocube Multifunctional Nanozyme with Oxygen Vacancies for Deep-Learning-Assisted Smartphone Biosensing and Organic Pollutant Degradation.

Although the application of nanozymes has been widely studied, it is still a huge challenge to develop highly active and multifunctional nanozyme catalysts with a wider application prospect. Co3O4/CoFe2O4 hollow nanocubes (HNCs) with oxygen vacancies were proposed in this study, which had a porous oxide heterostructure with CoFe2O4 as the core and Co3O4 as the shell. The Co3O4/CoFe2O4 HNCs had three enzyme activities: peroxidase-like, oxidase-like, and catalase-like. Combining XPS depth profiling with density functional theory (DFT), the catalytic mechanism of peroxidase-like activity was explored in depth, which was mainly originated from ·OH produced by the synergistic effect between the outer oxygen and inner oxygen and electron transfer between Co and Fe. A colorimetry/smartphone dual sensing platform was designed based on the peroxidase-like activity. Especially, a multifunctional intelligent sensing platform based on deep learning-YOLO v3 algorithm-assisted smartphone was constructed to realize real-time and rapid in situ detection of l-cysteine, norfloxacin, and zearalenone. Surprisingly, the detection limit of norfloxacin was low at 0.015 µM, which was better than that of the newly published detection method in the field of nanozymes. Meanwhile, the detection mechanism of l-cysteine and norfloxacin was successfully investigated by in situ FTIR. In fact, it also showed outstanding applications in detecting l-cysteine in the food environment and norfloxacin in drugs. Furthermore, Co3O4/CoFe2O4 HNCs also could degrade 99.24% of rhodamine B, along with good reusability even after 10-cycle runs. Therefore, this work provided an in-depth understanding of the synergistic effect between the outer and inner oxygen in the reaction mechanism and an efficient method for establishing a deep-learning-assisted intelligent detection platform. In addition, this research also offered a good guideline for the further development and construction of nanozyme catalysts with multienzyme activities and multifunctional applications.