A CYP78As-small grain4-coat protein complex â ¡ pathway promotes grain size in rice.

CYP78A, a cytochrome P450 subfamily that includes rice (Oryza sativa L.) BIG GRAIN2 (BG2, CYP78A13) and Arabidopsis thaliana KLUH (KLU, CYP78A5), generate an unknown mobile growth signal (referred to as a CYP78A-derived signal) that increases grain (seed) size. However, the mechanism by which the CYP78A pathway increases grain size remains elusive. Here, we characterized a rice small grain mutant, small grain4 (smg4), with smaller grains than its wild type due to restricted cell expansion and cell proliferation in spikelet hulls. SMG4 encodes a multidrug and toxic compound extrusion (MATE) transporter. Loss of function of SMG4 causes smaller grains while overexpressing SMG4 results in larger grains. SMG4 is mainly localized to endoplasmic reticulum (ER) exit sites (ERESs) and partially localized to the ER and Golgi. Biochemically, SMG4 interacts with coat protein complex Ⅱ (COPⅡ) components (Sar1, Sec23, and Sec24) and CYP78As (BG2, GRAIN LENGTH 3.2 [GL3.2], and BG2-LIKE 1 [BG2L1]). Genetically, SMG4 acts, at least in part, in a common pathway with Sar1 and CYP78As to regulate grain size. In summary, our findings reveal a CYP78As-SMG4-COPⅡ regulatory pathway for grain size in rice, thus providing new insights into the molecular and genetic regulatory mechanism of grain size.

A regulatory loop establishes the link between the circadian clock and abscisic acid signaling in rice.

There is a close regulatory relationship between the circadian clock and the abscisic acid (ABA) signaling pathway in regulating many developmental processes and stress responses. However, the exact feedback regulation mechanism between them is still poorly understood. Here, we identified the rice (Oryza sativa) clock component PSEUDO-RESPONSE REGULATOR 95 (OsPRR95) as a transcriptional regulator that accelerates seed germination and seedling growth by inhibiting ABA signaling. We also found that OsPRR95 binds to the ABA receptor gene REGULATORY COMPONENTS OF ABA RECEPTORS10 (OsRCAR10) DNA and inhibits its expression. Genetic analysis showed OsRCAR10 acts downstream of OsPRR95 in mediating ABA responses. In addition, the induction of OsPRR95 by ABA partly required a functional OsRCAR10, and the ABA-responsive element-binding factor ABSCISIC ACID INSENSITIVE5 (OsABI5) bound directly to the promoter of OsPRR95 and activated its expression, thus establishing a regulatory feedback loop between OsPRR95, OsRCAR10, and OsABI5. Taken together, our results demonstrated that the OsRCAR10-OsABI5-OsPRR95 feedback loop modulates ABA signaling to fine-tune seed germination and seedling growth, thus establishing the molecular link between ABA signaling and the circadian clock.

Rice STOMATAL CYTOKINESIS DEFECTIVE2 regulates cell expansion by affecting vesicular trafficking in rice.

Vesicular trafficking plays critical roles in cell expansion in yeast and mammals, but information linking vesicular trafficking and cell expansion in plants is limited. Here, we isolated and characterized a rice (Oryza sativa) mutant, decreased plant height 1-1 (dph1-1), which exhibited a wide spectrum of developmental phenotypes, including reduced plant height and smaller panicles and grains. Cytological analysis revealed that limited cell expansion was responsible for the dph1-1 mutant phenotype compared to the wild-type. Map-based cloning revealed that DPH1 encodes a plant-specific protein, OsSCD2, which is homologous to Arabidopsis (Arabidopsis thaliana) STOMATAL CYTOKINESIS DEFECTIVE2 (SCD2). Subcellular localization revealed that OsSCD2 is associated with clathrin. Confocal microscopy showed that the dph1-1 mutant has defective endocytosis and post-Golgi trafficking. Biochemical and confocal data indicated that OsSCD2 physically interacts with OsSCD1 and that they are associated with intracellular structures that colocalize with microtubules. Furthermore, we found that cellulose synthesis was affected in the dph1-1 mutant, evidenced by reduced cellulose synthase gene accumulation at the transcript and protein levels, most likely resulting from an impaired localization pattern. Our results suggest that OsSCD2 is involved in clathrin-related vesicular trafficking with an important role in maintaining plant growth in rice.

GPA5 Encodes a Rab5a Effector Required for Post-Golgi Trafficking of Rice Storage Proteins.

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice (Oryza sativa) glutelin precursor accumulation5 (gpa5) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant-specific VAMP727-containing R-soluble N-ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm.

Microstructure, mechanical properties, and retentive forces of cobalt-chromium removable partial denture frameworks fabricated by selective laser melting followed by heat treatment.

STATEMENT OF PROBLEM: The effect of heat treatment on the microstructure and mechanical properties of cobalt-chromium (Co-Cr) removable partial denture (RPD) frameworks fabricated by selective laser melting (SLM) is not well understood. PURPOSE: The purpose of this in vitro study was to evaluate the suitability of SLM-fabricated Co-Cr alloys followed by heat treatment as a framework for RPDs by determining the microstructure and mechanical properties. MATERIAL AND METHODS: Dumbbell specimens and RPD frameworks were fabricated by using SLM followed by heat treatment. The effects of the heat treatment on the microstructure were studied by using optical microscopy, scanning electron microscopy (SEM), and X-ray diffraction (XRD). Tensile and insertion and removal tests were performed to study the mechanical responses of selective laser melting followed by heat treatment specimens, including the ultimate tensile strength (UTS), 0.2% yield strength (0.2% YS), elongation (E), and retentive forces. Specimens fabricated by using the traditional lost-wax process were used as a control (casting) group. RESULTS: X-ray diffraction indicated that the γ-face-centered cubic phase dominated SLM and selective laser melting followed by heat treatment specimens. Results from optical microscopy and SEM showed microstructural changes under different fabrication and postprocessing heat treatments; it was difficult to observe the grain boundary in the SLM group, whereas submicrometer-scale grains had formed in the selective laser melting followed by heat treatment group. The selective laser melting followed by heat treatment group exhibited the highest elongation and retentive forces compared with the casting and SLM groups. CONCLUSIONS: SLM increased the mechanical properties of Co-Cr alloys. Postprocessing heat treatment further enhanced the tensile ductility. It is suggested that SLM followed by heat treatment is an efficient strategy for fabricating RPD frameworks.

OsCNGC13 promotes seed-setting rate by facilitating pollen tube growth in stylar tissues.

Seed-setting rate is a critical determinant of grain yield in rice (Oryza sativa L.). Rapid and healthy pollen tube growth in the style is required for high seed-setting rate. The molecular mechanisms governing this process remain largely unknown. In this study, we isolate a dominant low seed-setting rate rice mutant, sss1-D. Cellular examination results show that pollen tube growth is blocked in about half of the mutant styles. Molecular cloning and functional assays reveals that SSS1-D encodes OsCNGC13, a member of the cyclic nucleotide-gated channel family. OsCNGC13 is preferentially expressed in the pistils and its expression is dramatically reduced in the heterozygous plant, suggesting a haploinsufficiency nature for the dominant mutant phenotype. We show that OsCNGC13 is permeable to Ca2+. Consistent with this, accumulation of cytoplasmic calcium concentration ([Ca2+]cyt) is defective in the sss1-D mutant style after pollination. Further, the sss1-D mutant has altered extracellular matrix (ECM) components and delayed cell death in the style transmission tract (STT). Based on these results, we propose that OsCNGC13 acts as a novel maternal sporophytic factor required for stylar [Ca2+]cyt accumulation, ECM components modification and STT cell death, thus facilitating the penetration of pollen tube in the style for successful double fertilization and seed-setting in rice.

DELAYED HEADING DATE1 interacts with OsHAP5C/D, delays flowering time and enhances yield in rice.

Heading date is an important agronomic trait affecting crop yield. The GRAS protein family is a plant-specific super family extensively involved in plant growth and signal transduction. However, GRAS proteins are rarely reported have a role in regulating rice heading date. Here, we report a GRAS protein DHD1 (Delayed Heading Date1) delays heading and enhances yield in rice. Biochemical assays showed DHD1 physically interacts with OsHAP5C/D both in vitro and in vivo. DHD1 and OsHAP5C/D located in the nucleus and showed that rhythmic expression. Both DHD1 and OsHAP5C/D affect heading date by regulating expression of Ehd1. We propose that DHD1 interacts with OsHAP5C/D to delay heading date by inhibiting expression of Ehd1.

Early heading 7 interacts with DTH8, and regulates flowering time in rice.

KEY MESSAGE: EH7/Ghd7 interacts with DTH8, and regulates heading date by controlling the expression of Ehd1 in rice. Heading date, or flowering time, an important agronomic trait, influences regional adaptability and yield of crops. Many genes related to heading date in rice have been identified, and a preliminary regulatory network has been established, but the relationships between proteins involved are poorly understood. We identified a flowering suppressor EH7 (Early heading 7) that represses flowering under long-day (LD) conditions. The eh7 allele caused earlier heading, shorter plant height and less grain per main panicle than did the wild type (WT), but the tiller number and 1000-grain weight were not significantly affected under natural long-day conditions. Biochemical assays showed that EH7 interacts with DTH8. Quantitative PCR showed that EH7 inhibited heading date by downregulating the expression of Ehd1, Hd3a and RFT1. We propose that EH7 interacts with DTH8 to control flowering time by regulating the expression of Ehd1, Hd3a and RFT1.

OsLBD37 and OsLBD38, two class II type LBD proteins, are involved in the regulation of heading date by controlling the expression of Ehd1 in rice.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins are plant-specific transcription factors which are involved in many biological process of plant development, including lateral organ development, photomorphogenesis, pathogen response, pollen development and plant regeneration. Here, we report new functions of LBD proteins that participate in the regulation of heading date and yield in rice. OsLBD37 and OsLBD38 are two class II type LBD proteins, function as transcriptional activators. Overexpression of OsLBD37 and OsLBD38 separately causes delayed heading date and increased yield. Both OsLBD37 and OsLBD38 are expressed in rhythmic pattern, and their proteins are localized in the nucleus. Further analysis revealed that OsLBD37 and OsLBD38 delayed heading date by down-regulating the expression of the florigen genes Hd3a and RFT1 through key regulator of heading date Ehd1. These data indicates that OsLBD37 and OsLBD38, may function as novel regulators of heading date and crop yield in rice.

The OsHAPL1-DTH8-Hd1 complex functions as the transcription regulator to repress heading date in rice.

Heading date is an important agronomic trait related to crop yield. Many genes related to heading date have already been identified in rice (Oryza sativa), and a complicated, preliminary regulatory genetic network has also already been established, but the protein regulatory network is poorly understood. We have identified a novel heading date regulator, Heme Activator Protein like 1 (OsHAPL1), which inhibits flowering under long-day conditions. OsHAPL1 is a nuclear-localized protein that is highly expressed in leaves in a rhythmic manner. OsHAPL1 can physically interact with Days To Heading on chromosome 8 (DTH8), which physically interacts with Heading date 1 (Hd1) both in vitro and in vivo. OsHAPL1 forms a complex with DTH8 and Hd1 in Escherichia coli. OsHAPL1, DTH8, and Hd1 physically interact with the HAP complex, and also with general transcription factors in yeast (Saccharomyces cerevisiae). Further studies showed that OsHAPL1 represses the expression of the florigen genes and FLOWERING LOCUS T 1 (RFT1) and Hd3a through Early heading date 1 (Ehd1). We propose that OsHAPL1 functions as a transcriptional regulator and, together with DTH8, Hd1, the HAP complex, and general transcription factors, regulates the expression of target genes and then affects heading date by influencing the expression of Hd3a and RFT1 through Ehd1.

A CONSTANS-like transcriptional activator, OsCOL13, functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1 in rice.

Flowering time determines the adaptability of crop plants to different local environments, thus being one of the most important agronomic traits targeted in breeding programs. Photoperiod is one of the key factors that control flowering in plant. A number of genes that participate in the photoperiod pathway have been characterized in long-day plants such as Arabidopsis, as well as in short-day plants such as Oryza sativa. Of those, CONSTANS (CO) as a floral integrator promotes flowering in Arabidopsis under long day conditions. In rice, Heading date1 (Hd1), a homologue of CO, functions in an opposite way, which inhibits flowering under long day conditions and induces flowering under short day conditions. Here, we show that another CONSTANS-like (COL) gene, OsCOL13, negatively regulates flowering in rice under both long and short day conditions. Overexpression of OsCOL13 delays flowering regardless of day length. We also demonstrated that OsCOL13 has a constitutive and rhythmic expression pattern, and that OsCOL13 is localized to the nucleus. OsCOL13 displays transcriptional activation activity in the yeast assays and likely forms homodimers in vivo. OsCOL13 suppresses the florigen genes Hd3a and RFT1 by repressing Ehd1, but has no relationship with other known Ehd1 regulators as determined by using mutants or near isogenic lines. In addition, the transcriptional level of OsCOL13 significantly decreased in the osphyb mutant, but remained unchanged in the osphya and osphyc mutants. Thus, we conclude that OsCOL13 functions as a negative regulator downstream of OsphyB and upstream of Ehd1 in the photoperiodic flowering in rice.

OsCOL10, a CONSTANS-Like Gene, Functions as a Flowering Time Repressor Downstream of Ghd7 in Rice.

Flowering time, or heading date, is a critical agronomic trait that determines the cropping season and regional adaptability, and ultimately grain yield in rice. A number of genes involved in photoperiodic flowering have been cloned and their roles in modulating expression of the flowering genes have been characterized to a certain extent. However, much less is known about the pathway in transmitting the day length response signal(s) to induce transition to reproductive growth. Here, we report a constitutive flowering repressor OsCOL10, which encodes a member of the CONSTANS-like (COL) family. Transgenic rice plants overexpressing OsCOL10 (driven by a strong promoter or by fusing it to the activation domain of VP64) showed delayed flowering time under both short and long days.OsCOL10 is affected by the circadian clock and is preferentially expressed in leaf mesophyll cells; it is localized to the nucleus and has transcriptional activation activity. Further studies show that OsCOL10 represses the expression of theFT-like genes RFT1 and Hd3a through Ehd1. Transcripts of OsCOL10 are more abundant in plants carrying a functional Ghd7 allele or overexpressing Ghd7 than in Ghd7-deficient plants, thus placing OsCOL10 downstream of Ghd7.Taking these findings together, we conclude that OsCOL10 functions as a flowering time repressor that links Ghd7 and Ehd1 in rice.

Spatiotemporal control of root immune responses during microbial colonization.

The entire evolutionary trajectory of plants towards large and complex multi-cellular organisms has been accompanied by incessant interactions with omnipresent unicellular microbes. This led to the evolution of highly complex microbial communities, whose members display the entire spectrum of pathogenic to mutualistic behaviors. Plant roots are dynamic, fractally growing organs and even small Arabidopsis roots harbor millions of individual microbes of diverse taxa. It is evident that microbes at different positions on a root surface could experience fundamentally different environments, which, moreover, rapidly change over time. Differences in spatial scales between microbes and roots compares to humans and the cities they inhabit. Such considerations make it evident that mechanisms of root-microbe interactions can only be understood if analyzed at relevant spatial and temporal scales. This review attempts to provide an overview of the rapid recent progress that has been made in mapping and manipulating plant damage and immune responses at cellular resolution, as well as in visualizing bacterial communities and their transcriptional activities. We further discuss the impact that such approaches will have for a more predictive understanding of root-microbe interactions.

An innovative modulating functions method for pseudo-state estimation of fractional order systems.

In this paper, the objective is to estimate the pseudo-state of fractional order systems defined by the Caputo fractional derivative from discrete noisy output measurement. For this purpose, an innovative modulating functions method is proposed, which can provide non-asymptotic estimation within finite-time and is robust against corrupting noises. First, the proposed method is directly applied to the Brunovsky's observable canonical form of the considered system. Then, the initial value of the pseudo-state is exactly expressed by an algebraic integral formula, based on which the pseudo-state is estimated. Second, the properties and construction of the required modulating functions are studied. Furthermore, error analysis is provided in discrete noise cases, which is useful for improving the estimation accuracy. In order to show the advantages of the proposed method, two numerical examples are given, where both rational order and irrational order dynamical systems are considered. After selecting the design parameters using the provided noise error bound, the pseudo-states of considered systems are estimated. The fractional order Luenberger-like observer and the fractional order H∞-like observer are also applied. Better than the applied fractional order observers, the proposed method can guarantee the convergence speed and robustness at the same time.

The improved analytical stochastic model of infiltration trenches for stormwater quantity control.

As one of the infiltration-based low-impact development (LID) measures, infiltration trenches are widely used to reduce runoff and improve water quality. The conventional analytical stochastic approach developed for use in the hydrologic design of infiltration trenches often overestimates the trench's runoff reduction performance when the infiltration rate at the bottom of the trench exceeds some high level or when the size of the trench is smaller than some threshold level. Furthermore, the appropriateness of using kernel density estimation (KDE) for rainfall event separation and frequency analysis has not been examined yet in the actual hydrologic design of LIDs. To overcome these deficiencies, an improved analytical stochastic model (ASM) was developed in this study incorporating the KDE-based rainfall event characterization and a modified formula for estimating the effective storage capacity of trenches. The calibration, verification and application of the improved ASM were systematically presented and their results were discussed. The accuracy of the improved ASM were verified by comparing the analytical results against the corresponding continuous simulation results. A large number of design cases in nine provincial capital cities of China were analyzed using the improved ASM and considering the effects of soil types, trench's storage reservoir depth, area ratio, and climate conditions. The improved ASM of infiltration trenches is useful for quickly and accurately assessing their water quantity control performances. The results indicated that the accuracy of improved ASM improved by up to 71 % in terms of R-square among the 9 study areas compared to conventional ASM. The improved ASM can be used to directly and quickly calculate the useful hydrologic performance indices for a given trench size, soil condition, area ratio and local climate condition, it can thus provide scientific guidance for the Sponge City construction in China and sustainable urban stormwater management.

Directionally-Dependent Mechanical Properties of Ti6Al4V Manufactured by Electron Beam Melting (EBM) and Selective Laser Melting (SLM).

An investigation of mechanical properties of Ti6Al4V produced by additive manufacturing (AM) in the as-printed condition have been conducted and compared with wrought alloys. The AM samples were built by Selective Laser Melting (SLM) and Electron Beam Melting (EBM) in 0°, 45° and 90°-relative to horizontal direction. Similarly, the wrought samples were also cut and tested in the same directions relative to the plate rolling direction. The microstructures of the samples were significantly different on all samples. α' martensite was observed on the SLM, acicular α on EBM and combination of both on the wrought alloy. EBM samples had higher surface roughness (Ra) compared with both SLM and wrought alloy. SLM samples were comparatively harder than wrought alloy and EBM. Tensile strength of the wrought alloy was higher in all directions except for 45°, where SLM samples showed higher strength than both EBM and wrought alloy on that direction. The ductility of the wrought alloy was consistently higher than both SLM and EBM indicated by clear necking feature on the wrought alloy samples. Dimples were observed on all fracture surfaces.

DHD4, a CONSTANS-like family transcription factor, delays heading date by affecting the formation of the FAC complex in rice.

Heading date (or flowering time) is one of the most important agronomic traits in rice, influencing its regional adaptability and crop yield. Many major-effect genes for rice heading date have been identified, but in practice they are difficult to be used for rice molecular breeding because of their dramatic effects on heading date. Genes with minor effects on heading date, which are more desirable for fine-tuning flowering time without significant yield penalty, were seldom reported. In this study, we identified a new minor-effect heading date repressor, Delayed Heading Date 4 (DHD4). The dhd4 mutant shows a slightly earlier flowering phenotype without a notable yield penalty compared with wild-type plants under natural long-day conditions. DHD4 encodes a CONSTANS-like transcription factor localized in the nucleus. Molecular, biochemical, and genetic assays show that DHD4 can compete with 14-3-3 to interact with OsFD1, thus affecting the formation of the Hd3a-14-3-3-OsFD1 tri-protein FAC complex, resulting in reduced expression of OsMADS14 and OsMADS15, and ultimately delaying flowering. Taken together, these results shed new light on the regulation of flowering time in rice and provide a promising target for fine-tuning flowering time to improve the regional adaptability of rice.

Transcriptional activation and phosphorylation of OsCNGC9 confer enhanced chilling tolerance in rice.

Low temperature is a major environmental factor that limits plant growth and productivity. Although transient elevation of cytoplasmic calcium has long been recognized as a critical signal for plant cold tolerance, the calcium channels responsible for this process have remained largely elusive. Here we report that OsCNGC9, a cyclic nucleotide-gated channel, positively regulates chilling tolerance by mediating cytoplasmic calcium elevation in rice (Oryza sativa). We showed that the loss-of-function mutant of OsCNGC9 is defective in cold-induced calcium influx and more sensitive to prolonged cold treatment, whereas OsCNGC9 overexpression confers enhanced cold tolerance. Mechanistically, we demonstrated that in response to chilling stress, OsSAPK8, a homolog of Arabidopsis thaliana OST1, phosphorylates and activates OsCNGC9 to trigger Ca2+ influx. Moreover, we found that the transcription of OsCNGC9 is activated by a rice dehydration-responsive element-binding transcription factor, OsDREB1A. Taken together, our results suggest that OsCNGC9 enhances chilling tolerance in rice through regulating cold-induced calcium influx and cytoplasmic calcium elevation.

A numerical approximation method for fractional order systems with new distributions of zeros and poles.

A multiple-zero-pole (MZP) method is proposed for general SISO fractional order dynamic systems in this paper. Based on amplitude-frequency curve, a new rational approach to fractional differentiator is designed. There are three advantages of MZP method. 1) A more generalized form of approximation system is proposed by design the distribution of zeros and poles in a new way. 2) The same fractional differentiator can be approximated in many different forms. 3) The robustness of the approximation system is enhanced by using integer order integrators to construct fractional differentiator. The feasibility of the method is assessed in the illustrative examples, and the simulations prove the effectiveness.

WRKY Transcription Factor OsWRKY29 Represses Seed Dormancy in Rice by Weakening Abscisic Acid Response.

For efficient plant reproduction, seed dormancy delays seed germination until the environment is suitable for the next generation growth and development. The phytohormone abscisic acid (ABA) plays important role in the induction and maintenance of seed dormancy. Previous studies have identified that WRKY transcription factors can regulate ABA signaling pathway. Here, we identified an Oswrky29 mutant with enhanced dormancy in a screen of T-DNA insertion population. OsWRKY29 is a member of WRKY transcription factor family which located in the nuclear. The genetic analyses showed that both knockout and RNAi lines of OsWRKY29 had enhanced seed dormancy whereas its overexpression lines displayed reduced seed dormancy. When treated with ABA, OsWRKY29 knockout and RNAi lines showed greater sensitivity than its overexpression lines. In addition, the expression levels of ABA positive response factors OsVP1 and OsABF1 were higher in the OsWRKY29 mutants but were lower in its overexpression lines. Further assays showed that OsWRKY29 could bind to the promoters of OsABF1 and OsVP1 to inhibit their expression. In summary, we identified a new ABA signaling repressor OsWRKY29 that represses seed dormancy by directly downregulating the expression of OsABF1 and OsVP1.