Microstructure-united heterogeneous sodium alginate doped injectable hydrogel for stable hemostasis in dynamic mechanical environments.

Injectable hydrogels that can withstand compressive and tensile forces hold great promise for preventing rebleeding in dynamic mechanical environments after emergency hemostasis of wounds. However, current injectable hydrogels often lack sufficient compressive or tensile performance. Here, a microstructure-united heterogeneous injectable hydrogel (MH) was constructed. The heterogeneous structure endowed MH with a unique "microstructures consecutive transmission" feature, which allowed it to exhibit high compressive and tensile performance simultaneously. In this work, two types of sodium alginate doped hydrogels with different microstructures were physically smashed into microgels, respectively. By mixing the microgels, MH with one micro-pores featured microstructure and another nano-pores featured microstructure can be formed. The obtained MH can withstand both compressive and tensile forces and showed high mechanical performance (compressive modulus: 345.67 ± 10.12 kPa and tensile modulus: 245.19 ± 7.82 kPa). Furtherly, MH was proven to provide stable and sustained hemostasis in the dynamic mechanical environment. Overall, this work provided an effective strategy for constructing injectable hydrogel with high compressive and tensile performance for hemostasis in dynamic mechanical environments.

Highly stretchable, injectable hydrogels with cyclic endurance and shape-stability in dynamic mechanical environments, by microunit reformation.

Traditional injectable hydrogels have so far found it difficult to accommodate resistance to large deformation and shape-stability under cyclic deformation. Polyampholyte (PA) hydrogels exhibit resistance to large deformation, good fatigue-resistance and rapid self-healing under dynamic forces. The limitations of the preparation process result in non-injectability of polyampholyte (PA) hydrogels. Electrostatic interactions as a medium for resistance to large deformation and shape-stability after cyclic deformation in reformed injectable hydrogels has been explored in this study. The prepared hydrogels (as-prepared PA-N) were dried and smashed into microunits and then mixed with 0.9% NaCl solution to transform them into reformed hydrogels (as-reformed PA-N) via a needle to achieve injectability. The as-reformed PA-N could exhibit 913.6% elongation at break and showed shape-stability under cyclic deformation due to the efficient self-healing abilities of the microunits and the inherited structure of the prepared hydrogels, which are superior to those of current tough injectable hydrogels. Potential applications in elbow cyclic bending and frequent movement of mobile wounds have been proved in this study. Overall, the results showed that the as-reformed PA-N achieved convenient injectability with resistance to large deformation and shape-stability under cyclic deformation at the same time.

RD29A promoter constitutively drives a rice Hd3a expression to promote early-flowering in Saussurea involucrate Kar. et Kir. ex Maxim.

S. involucratae, an endemic and endangered plant, is a valuable and traditional Chinese medicinal herb. In order to control the flowering time of S. involucratae, we used the well-known stress inducible RD29A promoter to drive Hd3a (a FT ortholog from rice) expression in S. involucratae. Unexpectedly, the majority of regenerated buds in RD29A::Hd3a transgenic lines (S-RH) produced flowers in tissue culture stage under normal growth (25 ± 2 °C) condition. Their flowering time was not further influenced by salt treatment. Hd3a in S-RH was strongly expressed in MS media supplemented with or without 50 mM NaCl. RD29A::GUS transgenic experiments further revealed that RD29A constitutively promoted GUS expression in both S. involucrate and halophyte Thellungiella halophile, in contrast to glycophic plants Oryza sativa L. 'Zhonghua 11', in which its expression was up-regulated by cold, salinity, and drought stress. The results supported the hypothesis that RD29A promoter activity is inducible in stress-sensitive plants, but constitutive in stress-tolerant ones. Importantly, S-RH plants produced pollen grains and seeds under normal conditions. Additionally, we found that OsLEA3-1::Hd3a and HSP18.2::Hd3a could not promote S. involucrate to flower under either normal conditions or abiotic stresses. Taken together, we demonstrated the potential of RD29A::Hd3a might be served as a feasible approach in breeding S. involucrate under normal condition.

Pyramiding of gn1a, gs3, and ipa1 Exhibits Complementary and Additive Effects on Rice Yield.

Pyramiding of quantitative trait loci (QTLs) is a powerful approach in breeding super-high-yield varieties. However, the performance of QTLs in improving rice yield varies with specific genetic backgrounds. In a previous study, we employed the CRISPR/Cas9 system to target three yield-related genes, gn1a, gs3, and ipa1 in japonica 'Zhonghua 11', mutants of which featured large panicle, big grain, few sterile tillers, and thicker culm, respectively. In this paper, four pyramided lines, including gn1a-gs3, gn1a-ipa1, gs3-ipa1, and gn1a-gs3-ipa1, were further generated by conventional cross-breeding to be tested. Agronomic traits analysis showed that: (1) the stacking lines carried large panicles with an increased spikelet number in the main panicle or panicle; (2) the grain weight of the stacking lines, especially gs3-ipa1 and gn1a-gs3-ipa1, were heavier than those in single mutants; (3) both gn1a-gs3 and gs3-ipa1 produced more grain yield per plant than single mutant lines; (4) pyramided lines were higher than single mutants and transcriptome analysis found improved expression levels of genes related to lipid, amino acid, and carbohydrate transport and metabolism in lines pyramiding three mutant alleles, possibly as a result of complementary and additive effects. Accordingly, the alteration of gene-expression patterns relating to hormone signaling, plant growth, and seed size control was characterized in pyramided lines. The present study not only investigates the effects of pyramiding genes, but also may provide an efficient strategy for breeding super-high-yield rice by reducing the time cost of developing pyramided lines.

Target Lines for in Planta Gene Stacking in Japonica Rice.

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded ~4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development.

WRINKLED1 (WRI1) is a transcription factor which is key to the regulation of seed oil biosynthesis in Arabidopsis. In the study, we identified two WRI1 genes in rice, named OsWRI1a and OsWRI1b, which share over 98% nucleotide similarity and are expressed only at very low levels in leaves and endosperms. The subcellular localization of Arabidopsis protoplasts showed that OsWRI1a encoded a nuclear localized protein. Overexpression of OsWRI1a under the control of the CaMV 35S promoter severely retarded plant growth and development in rice. Expressing the OsWRI1a gene under the control of the P1 promoter of Brittle2 (highly expressed in endosperm but low in leaves and roots) increased the oil content of both leaves and endosperms and upregulated the expression of several genes related to late glycolysis and fatty acid biosynthesis. However, the growth and development of the transgenic plants were also affected, with phenotypes including smaller plant size, later heading time, and fewer and lighter grains. The laminae (especially those of flag leaves) did not turn green and could not unroll normally. Thus, ectopic expression of OsWRI1a in rice enhances oil biosynthesis, but also leads to abnormal plant growth and development.

Roles of AGD2a in Plant Development and Microbial Interactions of Lotus japonicus.

Arabidopsis AGD2 (Aberrant Growth and Death2) and its close homolog ALD1 (AGD2-like defense response protein 1) have divergent roles in plant defense. We previously reported that modulation of salicylic acid (SA) contents by ALD1 affects numbers of nodules produced by Lotus japonicus, but AGD2's role in leguminous plants remains unclear. A combination of enzymatic analysis and biological characterization of genetic materials was used to study the function of AGD2 (LjAGD2a and LjAGD2b) in L. japonicus. Both LjAGD2a and LjAGD2b could complement dapD and dapE mutants of Escherichia coli and had aminotransferase activity in vitro. ljagd2 plants, with insertional mutations of LjAGD2, had delayed flowering times and reduced seed weights. In contrast, overexpression of LjAGD2a in L. japonicus induced early flowering, with increases in seed and flower sizes, but reductions in pollen fertility and seed setting rates. Additionally, ljagd2a mutation resulted in increased expression of nodulin genes and corresponding increases in infection threads and nodule numbers following inoculation with Rhizobium. Changes in expression of LjAGD2a in L. japonicus also affected endogenous SA contents and hence resistance to pathogens. Our results indicate that LjAGD2a functions as an LL-DAP aminotransferase and plays important roles in plant development. Moreover, LjAGD2a activates defense signaling via the Lys synthesis pathway, thereby participating in legume-microbe interaction.

Genome-Wide Identification, Expression Patterns and Sugar Transport of the Physic Nut SWEET Gene Family and a Functional Analysis of JcSWEET16 in Arabidopsis.

The Sugars Will Eventually be Exported Transporters (SWEET) family is a class of sugar transporters that play key roles in phloem loading, seed filling, pollen development and the stress response in plants. Here, a total of 18 JcSWEET genes were identified in physic nut (Jatropha curcas L.) and classified into four clades by phylogenetic analysis. These JcSWEET genes share similar gene structures, and alternative splicing of messenger RNAs was observed for five of the JcSWEET genes. Three (JcSWEET1/4/5) of the JcSWEETs were found to possess transport activity for hexose molecules in yeast. Real-time quantitative PCR analysis of JcSWEETs in different tissues under normal growth conditions and abiotic stresses revealed that most are tissue-specifically expressed, and 12 JcSWEETs responded to either drought or salinity. The JcSWEET16 gene responded to drought and salinity stress in leaves, and the protein it encodes is localized in both the plasma membrane and the vacuolar membrane. The overexpression of JcSWEET16 in Arabidopsis thaliana modified the flowering time and saline tolerance levels but not the drought tolerance of the transgenic plants. Together, these results provide insights into the characteristics of SWEET genes in physic nut and could serve as a basis for cloning and further functional analysis of these genes.

Ectopic Expression of JcCPL1, 2, and 4 Affects Epidermal Cell Differentiation, Anthocyanin Biosynthesis and Leaf Senescence in Arabidopsis thaliana.

The CAPRICE (CPC)-like (CPL) genes belong to a single-repeat R3 MYB family, whose roles in physic nut (Jatropha curcas L.), an important energy plant, remain unclear. In this study, we identified a total of six CPL genes (JcCPL1-6) in physic nut. The JcCPL3, 4, and 6 proteins were localized mainly in the nucleus, while proteins JcCPL1, 2, and 5 were localized in both the nucleus and the cytoplasm. Ectopic overexpression of JcCPL1, 2, and 4 in Arabidopsis thaliana resulted in an increase in root hair number and decrease in trichome number. Consistent with the phenotype of reduced anthocyanin in shoots, the expression levels of anthocyanin biosynthesis genes were down-regulated in the shoots of these three transgenic A. thaliana lines. Moreover, we observed that OeJcCPL1, 2, 4 plants attained earlier leaf senescence, especially at the late developmental stage. Consistent with this, the expression levels of several senescence-associated and photosynthesis-related genes were, respectively, up-regulated and down-regulated in leaves. Taken together, our results indicate functional divergence of the six CPL proteins in physic nut. These findings also provide insight into the underlying roles of CPL transcription factors in leaf senescence.

The role of endogenous thiamine produced via THIC in root nodule symbiosis in Lotus japonicus.

Thiamine is a pivotal primary metabolite which is indispensable to all organisms. Although its biosynthetic pathway has been well documented, the mechanism by which thiamine influences the legume-rhizobium symbiosis remains uncertain. Here, we used overexpressing transgenic plants, mutants and grafting experiments to investigate the roles played by thiamine in Lotus japonicus nodulation. ljthic mutants displayed lethal phenotypes and the defect could be overcome by supplementation of thiamine or by overexpression of LjTHIC. Reciprocal grafting between L. japonicus wild-type Gifu B-129 and ljthic showed that the photosynthetic products of the aerial part made a major contribution to overcoming the nodulation defect in ljthic. Overexpression of LjTHIC in Lotus japonicus (OE-LjTHIC) decreased shoot growth and increased the activity of the enzymes 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. OE-LjTHIC plants exhibited an increase in the number of infection threads and also developed more nodules, which were of smaller size but unchanged nitrogenase activity compared to the wildtype. Taken together, our results suggest that endogenous thiamine produced via LjTHIC acts as an essential nutrient provided by the host plant for rhizobial infection and nodule growth in the Lotus japonicus - rhizobium interaction.

Overexpression of OsFTL10 induces early flowering and improves drought tolerance in Oryza sativa L.

Flowering time control is critically important for the reproductive accomplishment of higher plants as floral transition can be affected by both environmental and endogenous signals. Flowering Locus T-like (FTL) genes are major genetic determinants of flowering in plants. In rice, 13 OsFTL genes have been annotated in the genome and amongst them, Hd3a (OsFTL2) and RFT1 (OsFTL3) have been studied extensively and their functions are confirmed as central florigens that control rice flowering under short day and long day environment, respectively. In this report, a rice OsFTL gene, OsFTL10, was characterized, and its function on flowering and abiotic stress was investigated. The expression level of OsFTL10 was high in young seedlings and shown to be induced by GA3 and drought stress. Overexpression of OsFTL10 resulted in earlier flowering in rice plants by up to 2 weeks, through up-regulation of the downstream gene OsMADS15. OsFTL10 also regulated Ehd1 and OsMADS51 through a feedback mechanism. The OsFTL10 protein was also detected in both nucleus and cytoplasm. Furthermore, yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) results show that OsFTL10 could interact with multiple 14-3-3s, suggesting that OsFTL10 might function in a similar way to Hd3a in promoting rice flowering by forming a FAC complex with 14-3-3, and OsFD1. Further experiments revealed that constitutive expression of OsFTL10 improved the drought tolerance of transgenic plants by stimulating the expression of drought responsive genes. These results suggest that rice FTL genes might function in flowering promotion and responses to environmental signals.

The Temperature-Dependent Retention of Introns in GPI8 Transcripts Contributes to a Drooping and Fragile Shoot Phenotype in Rice.

Attachment of glycosylphosphatidylinositols (GPIs) to the C-termini of proteins is one of the most common posttranslational modifications in eukaryotic cells. GPI8/PIG-K is the catalytic subunit of the GPI transamidase complex catalyzing the transfer en bloc GPI to proteins. In this study, a T-DNA insertional mutant of rice with temperature-dependent drooping and fragile (df) shoots phenotype was isolated. The insertion site of the T-DNA fragment was 879 bp downstream of the stop codon of the OsGPI8 gene, which caused introns retention in the gene transcripts, especially at higher temperatures. A complementation test confirmed that this change in the OsGPI8 transcripts was responsible for the mutant phenotype. Compared to control plants, internodes of the df mutant showed a thinner shell with a reduced cell number in the transverse direction, and an inhomogeneous secondary wall layer in bundle sheath cells, while many sclerenchyma cells at the tops of the main veins of df leaves were shrunken and their walls were thinner. The df plants also displayed a major reduction in cellulose and lignin content in both culms and leaves. Our data indicate that GPI anchor proteins play important roles in biosynthesis and accumulation of cell wall material, cell shape, and cell division in rice.

Characterization of miRNA profiles in the mammary tissue of dairy cattle in response to heat stress.

BACKGROUND: MicroRNAs (miRNAs) are a class of small noncoding RNAs that play important roles in the regulation of gene expression. However, the role of miRNAs in bovine mammary gland responses to heat stress is not well understood. RESULTS: In the present study, we performed a deep RNA sequencing analysis to identify miRNAs associated with the heat stress potential of the bovine mammary gland. We identified 27 miRNAs that were differentially expressed significantly between the mammary tissue of Holstein cattle heat stress and normal conditions. Twenty miRNAs had higher expression in the mammary tissue of heat-stressed Holstein cattle. The seven highest differentially expressed candidate miRNAs (bta-miR-21-5p, bta-miR-99a-5p, bta-miR-146b, bta-miR-145, bta-miR-2285 t, bta-miR-133a, and bta-miR-29c) identified by deep RNA sequencing were additionally evaluated by stem-loop qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNAs expression in the mammary gland of heat-stressed versus control were associated with the regulation of Wnt, TGF-ß, MAPK, Notch, and JAK-STAT. CONCLUSIONS: These data indicated that the differentially expressed miRNAs identified in this study may act as dominant regulators during heat stress. We might reduce heat stress damage of Holstein cows by up-regulating or down-regulating these differentially expressed miRNAs.

Overexpression of a Phosphate Starvation Response AP2/ERF Gene From Physic Nut in Arabidopsis Alters Root Morphological Traits and Phosphate Starvation-Induced Anthocyanin Accumulation.

Physic nut (Jatropha curcas L.) is highly tolerant of barren environments and a significant biofuel plant. To probe mechanisms of its tolerance mechanisms, we have analyzed genome-wide transcriptional profiles of 8-week-old physic nut seedlings subjected to Pi deficiency (P-) for 2 and 16 days, and Pi-sufficient conditions (P+) controls. We identified several phosphate transporters, purple acid phosphatases, and enzymes of membrane lipid metabolism among the 272 most differentially expressed genes. Genes of the miR399/PHO2 pathway (IPS, miR399, and members of the SPX family) showed alterations in expression. We also found that expression of several transcription factor genes was modulated by phosphate starvation stress in physic nut seedlings, including an AP2/ERF gene (JcERF035), which was down-regulated in both root and leaf tissues under Pi-deprivation. In JcERF035-overexpressing Arabidopsis lines both numbers and lengths of first-order lateral roots were dramatically reduced, but numbers of root hairs on the primary root tip were significantly elevated, under both P+ and P- conditions. Furthermore, the transgenic plants accumulated less anthocyanin but had similar Pi contents to wild-type plants under P-deficiency conditions. Expression levels of the tested genes related to anthocyanin biosynthesis and regulation, and genes induced by low phosphate, were significantly lower in shoots of transgenic lines than in wild-type plants under P-deficiency. Our data show that down-regulation of the JcERF035 gene might contribute to the regulation of root system architecture and both biosynthesis and accumulation of anthocyanins in aerial tissues of plants under low Pi conditions.

Heterogeneity in the expression and subcellular localization of POLYOL/MONOSACCHARIDE TRANSPORTER genes in Lotus japonicus.

Polyols can serve as a means for the translocation of carbon skeletons and energy between source and sink organs as well as being osmoprotective solutes and antioxidants which may be involved in the resistance of some plants to biotic and abiotic stresses. Polyol/Monosaccharide transporter (PLT) proteins previously identified in plants are involved in the loading of polyols into the phloem and are reported to be located in the plasma membrane. The functions of PLT proteins in leguminous plants are not yet clear. In this study, a total of 14 putative PLT genes (LjPLT1-14) were identified in the genome of Lotus japonicus and divided into 4 clades based on phylogenetic analysis. Different patterns of expression of LjPLT genes in various tissues were validated by qRT-PCR analysis. Four genes (LjPLT3, 4, 11, and 14) from clade II were expressed at much higher levels in nodule than in other tissues. Moreover, three of these genes (LjPLT3, 4, and 14) showed significantly increased expression in roots after inoculation with Mesorhizobium loti. Three genes (LjPLT1, 3, and 9) responded when salinity and/or osmotic stresses were applied to L. japonicus. Transient expression of GFP-LjPLT fusion constructs in Arabidopsis and Nicotiana benthamiana protoplasts indicated that the LjPLT1, LjPLT6 and LjPLT7 proteins are localized to the plasma membrane, but LjPLT2 (clade IV), LjPLT3, 4, 5 (clade II) and LjPLT8 (clade III) proteins possibly reside in the Golgi apparatus. The results suggest that members of the LjPLT gene family may be involved in different biological processes, several of which may potentially play roles in nodulation in this nitrogen-fixing legume.

Global gene expression analysis of the response of physic nut (Jatropha curcas L.) to medium- and long-term nitrogen deficiency.

Jatropha curcas L. is an important biofuel plant with excellent tolerance of barren environments. However, studies on the regulatory mechanisms that operate in this plant in response to nitrogen (N) shortage are scarce. In this study, genome-wide transcriptional profiles of the roots and leaves of 8-week old physic nut seedlings were analyzed after 2 and 16 days of N starvation. Enrichment results showed that genes associated with N metabolism, processing and regulation of RNA, and transport predominated among those showing alterations in expression. Genes encoding transporter families underwent major changes in expression in both roots and leaves; in particular, those with roles in ammonia, amino acid and peptide transport were generally up-regulated after long-term starvation, while AQUAPORIN genes, whose products function in osmoregulation, were down-regulated. We also found that ASPARA-GINASE B1 and SARCOSINE OXIDASE genes were up-regulated in roots and leaves after 2 and 16 d N starvation. Genes associated with ubiquitination-mediated protein degradation were significantly up-regulated. In addition, genes in the JA biosynthesis pathway were strongly activated while expression of those in GA signaling was inhibited in leaves. We showed that four major classes of genes, those with roles in N uptake, N reutilization, C/N ratio balance, and cell structure and synthesis, were particularly influenced by long-term N limitation. Our discoveries may offer clues to the molecular mechanisms that regulate N reallocation and reutilization so as to maintain or increase plant performance even under adverse environmental conditions.

The Phenylalanine Ammonia Lyase Gene LjPAL1 Is Involved in Plant Defense Responses to Pathogens and Plays Diverse Roles in Lotus japonicus-Rhizobium Symbioses.

Phenylalanine ammonia lyase (PAL) is important in the biosynthesis of plant secondary metabolites that regulate growth responses. Although its function is well-established in various plants, the functional significance of PAL genes in nodulation is poorly understood. Here, we demonstrate that the Lotus japonicus PAL (LjPAL1) gene is induced by Mesorhizobium loti infection and methyl-jasmonate (Me-JA) treatment in roots. LjPAL1 altered PAL activity, leading to changes in lignin contents and thicknesses of cell walls in roots and nodules of transgenic plants and, hence, to structural changes in roots and nodules. LjPAL1-knockdown plants (LjPAL1i) exhibited increased infection thread and nodule numbers and the induced upregulation of nodulin gene expression after M. loti infection. Conversely, LjPAL1 overexpression delayed the infection process and reduced infection thread and nodule numbers after M. loti inoculation. LjPAL1i plants also exhibited reduced endogenous salicylic acid (SA) accumulation and expression of the SA-dependent marker gene. Their infection phenotype could be partially restored by exogenous SA or Me-JA application. Our data demonstrate that LjPAL1 plays diverse roles in L. japonicus-rhizobium symbiosis, affecting rhizobial infection progress and nodule structure, likely by inducing lignin modification, regulating endogenous SA biosynthesis, and modulating SA signaling.

Molecular cloning and characterization of two ß-ketoacyl-acyl carrier protein synthase I genes from Jatropha curcas L.

The ß-ketoacyl-acyl carrier protein synthase I (KASI) is involved in de novo fatty acid biosynthesis in many organisms. Two putative KASI genes, JcKASI-1 and JcKASI-2, were isolated from Jatropha curcas. The deduced amino acid sequences of JcKASI-1 and JcKASI-2 exhibit around 83.8% and 72.5% sequence identities with AtKASI, respectively, and both contain conserved Cys-His-Lys-His-Phe catalytic active sites. Phylogenetic analysis indicated that JcKASI-2 belongs to a clade with several KASI proteins from dicotyledonous plants. Both JcKASI genes were expressed in multiple tissues, most strongly in filling stage seeds of J. curcas. Additionally, the JcKASI-1 and JcKASI-2 proteins were both localized to the plastids. Expressing JcKASI-1 in the Arabidopsis kasI mutant rescued the mutant's phenotype and restored the fatty acid composition and oil content in seeds to wild-type, but expressing JcKASI-2 in the Arabidopsis kasI mutant resulted in only partial rescue. This implies that JcKASI-1 and JcKASI-2 exhibit partial functional redundancy and KASI genes play a universal role in regulating fatty acid biosynthesis, growth, and development in plants.

Screening of Proximal and Interacting Proteins in Rice Protoplasts by Proximity-Dependent Biotinylation.

Proximity-dependent biotin identification (BioID), which detects physiologically relevant proteins based on the proximity-dependent biotinylation process, has been successfully used in different organisms. In this report, we established the BioID system in rice protoplasts. Biotin ligase BirAG was obtained by removing a cryptic intron site in the BirA∗ gene when expressed in rice protoplasts. We found that protein biotinylation in rice protoplasts increased with increased expression levels of BirAG. The biotinylation effects can also be achieved by exogenous supplementation of high concentrations of biotin and long incubation time with protoplasts. By using this system, multiple proteins were identified that associated with and/or were proximate to OsFD2 in vivo. Our results suggest that BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment in plant cell.

Overexpression of the Starch Phosphorylase-Like Gene (PHO3) in Lotus japonicus has a Profound Effect on the Growth of Plants and Reduction of Transitory Starch Accumulation.

Two isoforms of starch phosphorylase (PHO; EC 2.4.1.1), plastidic PHO1 and cytosolic PHO2, have been found in all plants studied to date. Another starch phosphorylase-like gene, PHO3, which is an ortholog of Chlamydomonas PHOB, has been detected in some plant lineages. In this study, we identified three PHO isoform (LjPHO) genes in the Lotus japonicus genome. Expression of the LjPHO3 gene was observed in all tissues tested in L. japonicus, and the LjPHO3 protein was located in the chloroplast. Overexpression of LjPHO3 in L. japonicus resulted in a drastic decline in starch granule sizes and starch content in leaves. The LjPHO3 overexpression transgenic seedlings were smaller, and showed decreased pollen fertility and seed set rate. Our results suggest that LjPHO3 may participate in transitory starch metabolism in L. japonicus leaves, but its catalytic properties remain to be studied.