FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

Highly flexible carbon nitride-polyethylene glycol-cellulose acetate film with photocatalytic antibacterial activity for fruit preservation.

Cellulose acetate film, as a biodegradable and biomass-derived material, has great potential applications in food packaging. However, the poor mechanical and antibacterial properties limit its applications. Herein, a highly flexible carbon nitride-polyethylene glycol-cellulose acetate (CN-PEG-CA) film was successfully prepared by combining graphitic carbon nitride (g-C3N4) photocatalyst with cellulose acetate (CA). The g-C3N4 enables the film with antibacterial activity, as a green photocatalyst. PEG softens the rigid polymer CA and crosslinks CA, PEG, and g-C3N4 together by hydrogen bonding, as a flexible crosslinker. X-ray diffractometer (XRD), scanning electron microscope (SEM), and Fourier transform infrared spectrum (FT-IR) characterizations confirmed the successful preparation of the CN-PEG-CA film. The mechanical property tests demonstrated that adding PEG increased the elongation at break of the film by about 4 times. The composite film had high antibacterial activity, and the bactericidal rates on Escherichia coli and Staphylococcus aureus were 99.98 % and 99.89 %, respectively. It effectively extended the shelf life of strawberries to 96 h and effectively maintained the quality of strawberries during storage. After 96 h, the weight loss rate of strawberries packaged with 15 % CN-PEG-CA film was 21.83 %, vitamin C content was 45.47 %, titratable acidity content was 0.89 %, and color, hardness and total soluble solids were well maintained. And biocompatibility test results showed that the film was safe and nontoxic. From the ecological and economic point of view, the highly flexible and biodegradable films with efficient photocatalytic antibacterial activity synthesized in this paper have great potential in the field of food packaging.

The transcriptional hub SHORT INTERNODES1 integrates hormone signals to orchestrate rice growth and development.

Plants have evolved sophisticated mechanisms to coordinate their growth and stress responses via integrating various phytohormone signaling pathways. However, the precise molecular mechanisms orchestrating integration of the phytohormone signaling pathways remain largely obscure. In this study, we found that the rice (Oryza sativa) short internodes1 (shi1) mutant exhibits typical auxin-deficient root development and gravitropic response, brassinosteroid (BR)-deficient plant architecture and grain size as well as enhanced abscisic acid (ABA)-mediated drought tolerance. Additionally, we found that the shi1 mutant is also hyposensitive to auxin and BR treatment but hypersensitive to ABA. Further, we showed that OsSHI1 promotes the biosynthesis of auxin and BR by activating the expression of OsYUCCAs and D11, meanwhile dampens ABA signaling by inducing the expression of OsNAC2, which encodes a repressor of ABA signaling. Furthermore, we demonstrated that 3 classes of transcription factors, AUXIN RESPONSE FACTOR 19 (OsARF19), LEAF AND TILLER ANGLE INCREASED CONTROLLER (LIC), and OsZIP26 and OsZIP86, directly bind to the promoter of OsSHI1 and regulate its expression in response to auxin, BR, and ABA, respectively. Collectively, our results unravel an OsSHI1-centered transcriptional regulatory hub that orchestrates the integration and self-feedback regulation of multiple phytohormone signaling pathways to coordinate plant growth and stress adaptation.

A deleterious Sar1c variant in rice inhibits export of seed storage proteins from the endoplasmic reticulum.

KEY MESSAGE: We identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm. Higher plants accumlate large amounts of seed storage proteins (SSPs). However, mechanisms underlying SSP trafficking are largely unknown, especially the ER-Golgi anterograde process. Here, we showed that a rice glutelin precursor accumulation13 (gpa13) mutant exhibited floury endosperm and overaccumulated glutelin precursors, which phenocopied the reported RNAi-Sar1abc line. Molecular cloning revealed that the gpa13 allele encodes a mutated Sar1c (mSar1c) with a deletion of two conserved amino acids Pro134 and Try135. Knockdown or knockout of Sar1c alone caused no obvious phenotype, while overexpression of mSar1c resulted in seedling lethality similar to the gpa13 mutant. Transient expression experiment in tobacco combined with subcellular fractionation experiment in gpa13 demonstrated that the expression of mSar1c affects the subcellular distribution of all Sar1 isoforms and Sec23c. In addition, mSar1c failed to interact with COPII component Sec23. Conversely, mSar1c competed with Sar1a/b/d to interact with guanine nucleotide exchange factor Sec12. Together, we identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm.

Fructose-6-phosphate-2-kinase/fructose-2,6-bisphosphatase regulates energy metabolism and synthesis of storage products in developing rice endosperm.

Starch accounts for about 80-85 % of the dry weight of grains and determines yield by impact on grain weight. And, the content and composition of starch also determine appearance, eating, cooking and nutritional quality of rice. By coordinating crucial reactions of the primary carbohydrate metabolism in all eukaryotes, fructose-2,6-bisphosphate (Fru-2,6-P2) is a traffic signal in metabolism. However, the metabolic regulation of starch in plant sink tissues by Fru-2,6-P2 remains unclear. Here we isolated rice mutant floury endosperm23 (flo23) which has opaque endosperm and anomalous compound starch grains (SGs). flo23 mutant grains had reduced contents of starch, lipids and proteins. Map-based cloning and genetic complementation experiments showed that FLO23 encodes a cytoplasmic Fructose-6-phosphate-2-kinase/Fructose-2,6-bisphosphatase (F2KP). Mutation of OsF2KP2 decreased Fru-2,6-P2 content in endosperm cells, leading to drastically reduced phosphoenolpyruvate (PEP) and pyruvate contents and disordered glycolysis and energy metabolism. The results imply that OsF2KP2 participates in the glycolytic pathway by providing precursors and energy for synthesis of grain storage compounds.

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development.

Most of the reported P-type pentatricopeptide repeat (PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22 (flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein. Mutation of FLO22 resulting in defective trans-splicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22 mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly up-regulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

Dissolved oxygen sensor based on the fluorescence quenching method with optimal modulation frequency.

Measurement of dissolved oxygen (DO) in liquid samples is of vital importance in both industrial and biomedical fields. In this paper, a DO sensor based on the fluorescence quenching method has been built. The measurement principle is based on fluorescence lifetime detection, which is indicated by the phase difference between an excitation light signal and a fluorescence signal. The nonlinear effect of the fluorescent material has been taken into consideration to obtain a more accurate fitting model. The performance of the system varying with the modulation frequency of excitation light signals is also reported. Modulation frequency mainly affects the sensitivity and phase resolution ratio of the system. The system at the optimized modulation frequency has a good degree of fitting with R2 value of 0.9981 and a small relative error of 0.79%. The study shows that this kind of sensor with optimal modulation frequency has good performance, which can be used in many important fields.

Plastidic pyruvate dehydrogenase complex E1 component subunit Alpha1 is involved in galactolipid biosynthesis required for amyloplast development in rice.

Starch accounts for over 80% of the total dry weight in cereal endosperm and determines the kernel texture and nutritional quality. Amyloplasts, terminally differentiated plastids, are responsible for starch biosynthesis and storage. We screened a series of rice mutants with floury endosperm to clarify the mechanism underlying amyloplast development and starch synthesis. We identified the floury endosperm19 (flo19) mutant which shows opaque of the interior endosperm. Abnormal compound starch grains (SGs) were present in the endosperm cells of the mutant. Molecular cloning revealed that the FLO19 allele encodes a plastid-localized pyruvate dehydrogenase complex E1 component subunit α1 (ptPDC-E1-α1) that is expressed in all rice tissues. In vivo enzyme assays demonstrated that the flo19 mutant showed decreased activity of the plastidic pyruvate dehydrogenase complex. In addition, the amounts of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were much lower in the developing flo19 mutant endosperm, suggesting that FLO19 participates in fatty acid supply for galactolipid biosynthesis in amyloplasts. FLO19 overexpression significantly increased seed size and weight, but did not affect other important agronomic traits, such as panicle length, tiller number and seed setting rate. An analysis of single nucleotide polymorphism data from a panel of rice accessions identified that the pFLO19L haplotype was positively associated with grain length, implying a potential application in rice breeding. In summary, our study demonstrates that FLO19 is involved in galactolipid biosynthesis which is essential for amyloplast development and starch biosynthesis in rice.

Rice FLOURY SHRUNKEN ENDOSPERM 5 Encodes a Putative Plant Organelle RNA Recognition Protein that Is Required for cis-Splicing of Mitochondrial nad4 Intron 1.

BACKGROUND: The sequences of several important mitochondrion-encoded genes involved in respiration in higher plants are interrupted by introns. Many nuclear-encoded factors are involved in splicing these introns, but the mechanisms underlying this splicing remain unknown. RESULTS: We isolated and characterized a rice mutant named floury shrunken endosperm 5 (fse5). In addition to having floury shrunken endosperm, the fse5 seeds either failed to germinate or produced seedlings which grew slowly and died ultimately. Fse5 encodes a putative plant organelle RNA recognition (PORR) protein targeted to mitochondria. Mutation of Fse5 hindered the splicing of the first intron of nad4, which encodes an essential subunit of mitochondrial NADH dehydrogenase complex I. The assembly and NADH dehydrogenase activity of complex I were subsequently disrupted by this mutation, and the structure of the mitochondria was abnormal in the fse5 mutant. The FSE5 protein was shown to interact with mitochondrial intron splicing factor 68 (MISF68), which is also a splicing factor for nad4 intron 1 identified previously via yeast two-hybrid (Y2H) assays. CONCLUSION: Fse5 which encodes a PORR domain-containing protein, is essential for the splicing of nad4 intron 1, and loss of Fse5 function affects seed development and seedling growth.