Multi-genome evolutionary study of the ABC1 gene family and identification of the pleiotropic effects of OsABC1-13 in rice development

In four rice genomes,85 ABC1-family genes were identified by comparative genomics,evolution,genetics,and physiology.One,OsABC1-13,was shown by knockdown and knockout experiments to affect plant height,grain size,and photosynthetic capability.

OstMAPKKK5, a truncated mitogen-activated protein kinase kinase kinase 5, positively regulates plant height and yield in rice

Rice(Oryza sativa L.)is a major food crop worldwide.Plant height and yield are important agronomic traits of rice.Several genes regulating plant height and/or yield have been cloned.However,the molecular mechanisms coordinating plant height and yield are not fully characterized.Here,we report a novel gene,OstMAPKKK5 that encodes a truncated variant of a mitogen-activated protein kinase kinase kinase 5(OsMAPKKK5)lacking an intact kinase domain.Transgenic plants overexpressing OstMAPKKK5 in indica cultivar 9311 showed increased plant height,grain length,grain width,1000-grain weight,grain number per main panicle,and yield.Real-time quantitative PCR showed that OstMAPKKK5 was widely expressed in various tissues and developmental stages.The increased plant height and yield were attributed to enlarged cell size.Overexpression of OstMAPKKK5 led to higher contents of various forms of endogenous gibberellin(GA),especially the most common active forms,GA1,GA3,GA4.We concluded that OstMAPKKK5 positively regulates plant height and yield in rice by affecting cell size,and that its underlying mechanism is based on increased endogenous GA content.

TATA-box binding protein-associated factor 2 regulates grain size in rice

Grain size,characterized by a combination of grain length,width,and thickness,is one of the major determinants of yield in rice.The present study identified TATA-box binding protein-associated factor 2(TAF2)as an essential component regulating transcription and determining grain size in rice.Map-based cloning showed that a G/T substitution in TAF2 resulted in a naturally occurring mutant called reduced grain size and plant height 1(rgh1).The mutants,with weak edited rgh1 alleles,exhibited a small grain phenotype with reduced grain length and width,while the severe knockout mutant(rgh1-2s)was dwarf and completely sterile.Allelic test performed between rgh1 and several edited alleles confirmed that the mutation in TAF2 caused the rgh1 phenotype.GUS staining showed that TAF2 was mainly expressed in the vascular bundles of roots,stems,leaves,and grains.The cytological analysis revealed that reduced cell division in the glumes resulted in the small grain phenotype of rgh1.Further RNA-sequencing detected altered expression of genes involved in the basic biological processes in rgh1 mutant.These findings provide novel insights into the TAF2-mediated genetic mechanism regulating grain size in rice.

Post-transcriptional regulation of grain weight and shape by the RBP-A-J-K complex in rice

RNA-binding proteins(RBPs)are components of the post-transcriptional regulatory system,but their regulatory effects on complex traits remain unknown.Using an integrated strategy involving map-based cloning,functional characterizations,and transcriptomic and population genomic analyses,we revealed that RBP-K(LOC_Os08g23120),RBP-A(LOC_Os11g41890),and RBP-J(LOC_Os10g33230)encode proteins that form an RBP-A-J-K complex that negatively regulates rice yield-related traits.Examinations of the RBP-A-J-K complex indicated RBP-K functions as a relatively non-specific RBP chaperone that enables RBP-A and RBP-J to function normally.Additionally,RBP-J most likely affects GA pathways,resulting in considerable increases in grain and panicle lengths,but decreases in grain width and thickness.In contrast,RBP-A negatively regulates the expression of genes most likely involved in auxin-regulated pathways controlling cell wall elongation and carbohydrate transport,with substantial effects on the rice grain filling process as well as grain length and weight.Evolutionarily,RBP-K is relatively ancient and highly conserved,whereas RBP-J and RBP-A are more diverse.Thus,the RBP-A-J-K complex may represent a typical functional model for many RBPs and protein complexes that function at transcriptional and post-transcriptional levels in plants and animals for increased functional consistency,efficiency,and versatility,as well as increased evolutionary potential.Our results clearly demonstrate the importance of RBP-mediated post-transcriptional regulation for the diversity of complex traits.Furthermore,rice grain yield and quality may be enhanced by introducing various complete or partial loss-of-function mutations to specific RBP genes using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9 technology and by exploiting desirable natural tri-genic allelic combinations at the loci encoding the components of the RBP-A-J-K complex through marker-assisted selection.

A Novel QTL qTGW3 Encodes the GSK3/ SHAGGY-Like Kinase OsGSK5/OsSK41 that Interacts with OsARF4 to Negatively Regulate Grain Size and Weight in Rice

Grain size and shape are important determinants of grain weight and yield in rice. Here, we report a new major quantitative trait locus （QTL）, qTGW3, that controls grain size and weight in rice. This locus, qTGW3, encodes OsSK41 （also known as OsGSK5）, a member of the GLYCOGEN SYNTHASE KINASE 3/SHAGGY-like family. Rice near-isogenic lines carrying the loss-of-function allele of OsSK41 have increased grain length and weight. We demonstrate that OsSK41 interacts with and phosphorylates AUXIN RESPONSE FACTOR 4 （OsARF4）. Co-expression of OsSK41 with OsARF4 increases the accumulation of OsARF4 in rice protoplasts. Loss of function of OsARF4 results in larger rice grains. RNA-sequencing analysis suggests that OsARF4 and OsSK41 repress the expression of a common set of downstream genes, including some auxin-responsive genes, during rice grain development. The loss-of-function form of OsSK41 at qTGW3 represents a rare allele that has not been extensively utilized in rice breeding. Suppression of OsSK41 function by either targeted gene editing or QTL pyramiding enhances rice grain size and weight. Thus, our study reveals the important role of OsSK41 in rice grain development and provides new candidate genes for genetic improvement of grain yield in rice and perhaps in other cereal crops.

A Kelch Motif-Containing Serine/Threonine Protein Phosphatase Determines the Large Grain QTL Trait in Rice

A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice （Oryza sativa L.） varieties. In this study, we generated an F2 population by crossing the large-grain japonica cultivar CW23 with Peiai 64 （PA64）, an elite indica small-grain cultivar. Using QTL analysis, 17 QTLs for five grain traits were detected on four different chromosomes. Eight of the QTLs were newly-identified in this study. In particular, qGL3-1, a newly-identified grain length QTL with the highest LOD value and largest phenotypic variation, was fine-mapped to the 17 kb region of chromosome 3. A serine/threonine protein phosphatase gene encoding a repeat domain containing two Kelch motifs was identified as the unique candidate gene corresponding to this QTL. A comparison of PA64 and CW23 sequences revealed a single nucleotide substitution （C→A） at position 1092 in exon 10, resulting in replacement of Asp （D） in PA64 with Glu （E） in CW23 for the 364th amino acid. This variation is located at the D position of the conserved sequence motif AVLDT of the Kelch repeat. Genetic analysis of a near-isogenic line （NIL） for qGL3-1 revealed that the allele qGL3-1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker-assisted selection.

Additive and Over-dominant Effects Resulting from Epistatic Loci Are the Primary Genetic Basis of Heterosis in Rice

A set of 148 F9 recombinant inbred lines （RILs） was developed from the cross of an indica cultivar 93-11 and japonica cultivar DTI13, showing strong F1 heterosis. Subsequently, two backcross F1 （BCFI） populations were constructed by backcrossing these 148 RILs to two parents, 93-11 and DT713. These three related populations （281BCF1 lines, 148 RILs） were phenotyped for six yield-related traits in two locations. Significant inbreeding depression was detected in the population of RILS and a high level of heterosis was observed in the two BCF1 populations. A total of 42 main-effect quantitative trait loci （M-QTLs） and 109 epistatic effect QTL pairs （E-QTLs） were detected in the three related populations using the mixed model approach. By comparing the genetic effects of these QTLs detected in the RILs, BCF1 performance and mid-parental heterosis （HMp）, we found that, in both BCF1 populations, the QTLs detected could be classified into two predominant types： additive and over-dominant loci, which indicated that the additive and over-dominant effect were more important than complete or partially dominance for M-QTLs and E-QTLs. Further, we found that the E-QTLs detected collectively explained a larger portion of the total phenotypic variation than the M-QTLs in both RILs and BCF1 populations. All of these results suggest that additive and over-dominance resulting from epistatic loci might be the primary genetic basis of heterosis in rice.

Continuous monitoring of diabetes with an integrated microneedle biosensing device through 3D printing

Diabetes is a prevalent chronic metabolic disease with multiple clinical manifestations and complications,and it is among the leading causes of death.Painless and continuous monitoring of interstitial glucose is highly desirable for diabetes management.Here we unprecedentedly show continuous monitoring of diabetes with an integrated microneedle biosensing device.The device was manufactured with a 3D printing process,a microfabrication process,an electroplating process,and an enzyme immobilization step.The device was inserted into the dermis layer of mouse skin and showed accurate sensing performance for monitoring subcutaneous glucose levels in normal or diabetic mice.The detection results were highly correlated with those obtained from a commercial blood glucose meter.We anticipate that the study could open exciting avenues for monitoring and managing diabetes,alongside fundamental studies of subcutaneous electronic devices.

Auxin signaling module OsSK41-OsIAA10-OsARF regulates grain yield traits in rice

Auxin is an important phytohormone in plants, and auxin signaling pathways in rice play key roles in regulating its growth, development, and productivity. To investigate how rice grain yield traits are regulated by auxin signaling pathways and to facilitate their application in rice improvement, we validated the functional relationships among regulatory genes such as OsIAA10, OsSK41, and OsARF21 that are involved in one of the auxin(OsIAA10) signaling pathways. We assessed the phenotypic effects of these genes on several grain yield traits across two environments using knockout and/or overexpression transgenic lines.Based on the results, we constructed a model that showed how grain yield traits were regulated by OsIAA10 and OsTIR1, OsAFB2, and OsSK41 and OsmiR393 in the OsSK41-OsIAA10-OsARF module and by OsARF21 in the transcriptional regulation of downstream auxin response genes in the OsSK41-OsIAA10-OsARF module. The population genomic analyses revealed rich genetic diversity and the presence of major functional alleles at most of these loci in rice populations. The strong differentiation of many major alleles between Xian/indica and Geng/japonica subspecies and/or among modern varieties and landraces suggested that they contributed to improved productivity during evolution and breeding. We identified several important aspects associated with the genetic and molecular bases of rice grain and yield traits that were regulated by auxin signaling pathways.We also suggested rice auxin response factor(OsARF) activators as candidate target genes for improving specific target traits by overexpression and/or editing subspecies-specific alleles and by searching and pyramiding the ‘best' gene allelic combinations at multiple regulatory genes in auxin signaling pathways in rice breeding programs.

Genetic Analysis and Fine Mapping of a Novel Semidominant Dwarfing Gene LB4D in Rice

A dwarf mutant,designated LB4D,was obtained among the progeny of backcrosses to a wild rice introgression line.Genetic analysis of LB4D indicated that the dwarf phenotype was controlled by a single semidominant dwarfing gene,which was named LB4D.The mutants were categorized as dn-type dwarf mutants according to the pattern of internode reduction.In addition,gibberellin（GA） response tests showed that LB4D plants were neither deficient nor insensitive to GA.This study found that tiller formation by LB4D plants was decreased by 40%compared with the wild type,in contrast to other dominant dwarf mutants that have been identified,indicating that a different dwarfing mechanism might be involved in the LB4D dominant mutant.The reduction of plant height in F1 plants ranged from 27.9%to 38.1%in different genetic backgrounds,showing that LB4D exerted a stronger dominant dwarfing effect. Using large F2 and F3 populations derived from a cross between heterozygous LB4D and the japonica cultivar Nipponbare,the LB4D gene was localized to a 46 kb region between the markers Indel 4 and Indel G on the short arm of chromosome 11,and four predicted genes were identified as candidates in the target region.

The kinase OsSK41/OsGSK5 negatively regulates amylose content in rice endosperm by affecting the interaction between OsEBP89 and OsBP5

Amylose content(AC) is the main factor determining the palatability, viscosity, transparency, and digestibility of rice(Oryza sativa)grains. AC in rice grains is mainly controlled by different alleles of the Waxy(Wx) gene. The AP2/EREBP transcription factor OsEBP89 interacts with the MYC-like protein OsBP5 to synergistically regulate the expression of Wx.Here, we determined that the GLYCOGEN SYNTHASE KINASE 5(OsGSK5, also named SHAGGY-like kinase 41 [OsSK41]) inhibits the transcriptional activation activity of OsEBP89 in rice grains during amylose biosynthesis. The loss of OsSK41 function enhanced Wx expression and increased AC in rice grains. By contrast, the loss of function of OsEBP89 reduced Wx expression and decreased AC in rice grains. OsSK41 interacts with OsEBP89 and phosphorylates four of its sites(Thr-28,Thr-30, Ser-238, and Thr-257), which makes OsEBP89 unstable and attenuates its interaction with OsBP5. Wx promoter activity was relatively weak when regulated by the phosphomimicvariantOsEBP89E–OsBP5but relatively strong when regulated by the nonphosphorylatable variant OsEBP89A–OsBP5.Therefore, OsSK41-mediated phosphorylation of OsEBP89 represents an additional layer of complexity in the regulation of amylose biosynthesis during rice grain development. In addition, our findings provide four possible sites for regulating rice grain AC via precise gene editing.

A Microtube-Based Wearable Closed-Loop Minisystem for Diabetes Management

Diabetes is a chronic metabolic disease with a high blood glucose level,leading to both seriously acute and chronic complications.The closed-loop system is an ideal system for diabetes management.However,the large size and high cost of the commercial systems restrict their widespread uses.Here,we present for the first time a microtube-based wearable closed-loop minisystem for diabetes management.The closed-loop minisystem includes a biosensing device,an electroosmotic micropump,and a printed circuit board(PCB)with an algorithm.The microtube-based sensing device coated on the outer surface of the microtube is inserted into subcutaneous tissue for detecting interstitial glucose;the current signal for sensing glucose is processed by the PCB to power the electroosmotic micropump intelligently for the delivery of insulin into the subcutaneous tissue via the microtube channel.The closed-loop minisystem worn on a diabetic SD rat can successfully maintain its blood glucose level within a safe level.It is expected that this new closed-loop paradigm could open up new prospects for clinical diabetes management.