DENSE AND ERECT PANICLE 1 Research Articles

INHBA regulates Hippo signaling to confer 5-FU chemoresistance mediated by cellular senescence in colon cancer cells

Colon cancer has become a global public health challenge, and 5-Fluorouracil (5-FU) chemoresistance is a major obstacle in its treatment. Chemoresistance can be mediated by therapy-induced cellular senescence. This study intended to investigate mechanisms of INHBA (inhibin A) in 5-FU resistance mediated by cellular senescence in colon cancer. Bioinformatics analysis of INHBA expression in colon cancer tissues, survival analysis, and correlation analysis of cellular senescence markers were performed. The effects of INHBA on the biological characteristics and 5-FU resistance of colon cancer cells were examined through loss/gain-of-function and molecular assays. Finally, a xenograft mouse model was built to validate the mechanism of INHBA in vivo. INHBA was upregulated in colon cancer and was significantly positively correlated with cellular senescence markers uncoupling protein 2 (UCP-2), matrix metalloproteinase-1 (MMP-1), dense and erect panicle 1 (DEP1), and p21. Cellular senescence in colon cancer mediated 5-FU resistance. Downregulation of INHBA expression enhanced 5-FU sensitivity in colon cancer cells, inhibited cell proliferation, promoted apoptosis, increased the proportion of cells in G0/G1 phase, and it resulted in a lower proportion of senescent cells and lower levels of the cellular senescence markers interleukin 6 (IL-6) and interleukin 8 (IL-8). Analysis of whether to use the pathway inhibitor Verteporfin proved that INHBA facilitated colon cancer cell senescence and enhanced 5-FU chemoresistance via inactivation of Hippo signaling pathway, and consistent results were obtained in vivo. Collectively, INHBA conferred 5-FU chemoresistance mediated by cellular senescence in colon cancer cells through negative regulation of Hippo signaling.

Agronomic potential of plant-specific Gγ proteins.

The vascular plant-specific type III Gγ proteins have emerged as important targets for biotechnological applications. These proteins are exemplified by Arabidopsis AGG3, rice Grain Size 3 (GS3), Dense and Erect Panicle 1 (DEP1), and GGC2 and regulate plant stature, seed size, weight and quality, nitrogen use efficiency, and multiple stress responses. These Gγ proteins are an integral component of the plant heterotrimeric G-protein complex and differ from the canonical Gγ proteins due to the presence of a long, cysteine-rich C-terminal region. Most cereal genomes encode three or more of these proteins, which have similar N-terminal Gγ domains but varying lengths of the C-terminal domain. The C-terminal domain is hypothesized to give specificity to the protein function. Intriguingly, many accessions of cultivated cereals have natural deletion of this region in one or more proteins, but the mechanistic details of protein function remain perplexing. Distinct, sometimes contrasting, effects of deletion of the C-terminal region have been reported in different crops or under varying environmental conditions. This review summarizes the known roles of type III Gγ proteins, the possible action mechanisms, and a perspective on what is needed to comprehend their full agronomic potential.

Heterotrimeric G protein γ subunit DEP1 is involved in hydrogen peroxide signaling and promotes aerenchyma formation in rice roots

ABSTRACT Heterotrimeric G-protein α and β-subunits regulate H2O2-mediated aerenchyma formation. The rice G-protein γ-subunit, dense and erect panicle 1 (DEP1), is known to interact with the α-subunit and regulate nitrogen utilization and yield. However, it is unclear whether DEP1 regulates cell death for aerenchyma formation in rice roots. Using wild-type WYJ8 and its transgenic line WYJ8(DEP1), we confirmed that DEP1 is involved in H2O2-mediated aerenchyma formation. The rates of aerenchyma formation varied in different parts of the roots in both varieties, with the highest rate in the 4–7 cm segments, reaching a plateau in the 7–8 cm segments. Compared with WYJ8, the aerenchyma area and H2O2 content in WYJ8(DEP1) were increased by 55.98% and 53.37%, respectively; however, the responses of aerenchyma formation to exogenous H2O2 were basically the same in the two varieties. Diphenylene iodonium (DPI) treatment had no effect on H2O2 production and elimination processes in WYJ8, but significantly reduced the activity of the key enzyme that catalyzes H2O2 biosynthesis in WYJ8(DEP1). Importantly, exogenous H2O2 treatment did not offset the effect of the decrease in endogenous H2O2 level caused by DPI on aerenchyma formation. These results indicated that DEP1 enhanced H2O2 biosynthesis and promoted the cell death of the root cortex, thus contributing to aerenchyma development in WYJ8(DEP1).

Effects of Genetic Background and Environmental Conditions on Amylopectin Chain-Length Distribution in a Recombinant Inbred Line of an Inter-subspecies Rice Cross.

Amylopectin is an essential starch property, and the chain-length distribution of amylopectin (APCLD) is closely associated with the eating and cooking quality of rice. In this study, a series of recombinant inbred lines derived from an indica/japonica cross were planted in four areas with distinct ecological conditions (LN, SC, JS, and GD), and the relationship among APCLD, environmental factors, and genetic background was analyzed. The results showed that APCLD was strongly influenced by environmental factors, which dynamically changed from heading to the mature stage. The solar radiation, luminous flux, and light hours were positively correlated with Fa but negatively correlated with Fb1 and Fb2. The temperature was negatively correlated with Fa and Fb1 but positively correlated with Fb2 and Fb3. The temperature was the primary factor affecting APCLD, followed by humidity and light. There was no significant correlation between the indica pedigree percentage and APCLD. Furthermore, we detected six quantitative trait loci related to Fa, Fb1, Fb2, and Fb3 chains, several of which shared a similar region to previously reported loci, including DENSE AND ERECT PANICLE 1 (DEP1). The truncated dep1 allele increased Fa, Fb2, and Fb3 but decreased Fb1 in LN, whereas Fa was decreased but Fb1 and Fb2 were increased in JS. Elucidating the effects of climate factors and genetic background on APCLD could provide a theoretical basis and technical guidance for high-quality rice breeding.

Identification of Multiple Grain Shape-Related Loci in Rice Using Bulked Segregant Analysis With High-Throughput Sequencing.

Grain shape (GS) is an important agronomic trait that can improve rice breeding for optimal appearance quality, and it varies highly between indica and japonica subspecies. In this study, we conducted a genome sequencing of a series of recombination inbred lines (RILs) derived from a cross between japonica variety Shennong265 (SN265) and indica variety R99, and we successfully detected seven loci associated with GS. Subsequent analysis showed that a major quantitative trait locus (QTL) corresponded to the qSW5/GW5. To identify a main-effect locus, we conducted bulked segregant analysis (BSA) in two F2 populations. A 0.8-Mb region on chromosome 9 was identified as the candidate region of GS. There are 101 predicted genes in this region, and eight single nucleotide polymorphisms/insertions and deletions (SNPs/INDELs) caused frameshift. We found that a 637-bp stretch in exon 5 of the DENSE AND ERECT PANICLE 1 (DEP1) locus in SN265 was replaced by a 12-bp sequence. The two types of CRISPR/Cas9 gene-edited plants confirmed that DEP1 affected GS, and both Gγ and Cys-rich domains participated in this regulatory mechanism. These findings improve our understanding of the underlying mechanism of GS in rice and provide an effective and rapid strategy for the identification of main-effect loci of target traits.

Effects of DEP1 on grain yield and grain quality in the background of two japonica rice (Oryza sativa) cultivars

AbstractPlant architecture is an accessible approach to achieving high‐yield potential. The DENSE AND ERECT PANICLE 1 (DEP1) gene regulating panicle morphology, grain number per panicle, and nitrogen uptake and metabolism has been widely used for the breeding of high‐yield rice in northern Chinese japonica varieties. However, there has been no consensus on the genetic effects of dep1 on grain yield and quality under different genetic backgrounds and growing environments. In the present study, we developed two sets of near‐isogenic lines (NILs) of DEP1 (AKI‐dep1, AKI‐DEP1, LG5‐dep1 and LG5‐DEP1), each carrying the DEP1 region from either 'Liaogeng5' (LG5) or 'Akitakomachi' (AKI) in the AKI and LG5 backgrounds. Our results demonstrated that AKI‐dep1/LG5‐dep1 exhibited erect panicle and enhanced grain number per panicle, thereby consequently increasing grain yield, whereas they possessed inferior grain appearance compared with AKI‐DEP1/LG5‐DEP1 in the same background. However, the effects of dep1 on grain processing quality and eating and cooking quality varied with the background. These results provide useful information for high‐yield erect panicle rice breeding by marker‐assisted selection.

Erect panicle architecture contributes to increased rice production through the improvement of canopy structure

The erect panicle architecture has contributed to the improvement of yield in japonica rice breeding, and recent molecular analysis has revealed the mechanisms involved in individual plant yield increases. However, the population structure is more important in rice production compared with individual plant yield. Our study compared the population canopy structure of a curved panicle variety Sasanishiki (WT) and an erect panicle mutant derived from CRISPR/Cas9 gene editing at the DENSE AND ERECT PANICLE 1 (DEP1) locus. The results showed that more light could reach to the leaves under the panicle in the CRISPR-dep1 population compared with the WT. The canopy of the CRISPR-dep1 population exhibited higher temperature and lower humidity compared with the WT after heading. A subsequent survey showed that the CO2 concentration in the CRISPR-dep1 population was significantly lower than that in the WT population from full heading to 15 days after heading. Moreover, the increase of biomass in the CRISPR-dep1 population was greater than that in the WT. We noticed that the CRISPR-dep1 mutant could achieve higher yield under low fertilization application compared with the WT under high fertilizer application through increased transplant density. These traits could contribute to an agricultural sustainable development strategy. The quality investigation showed that the dep1 allele increased the yield along with imposing a penalty on grain quality. Our study not only elucidated the mechanism of yield improvement in an erect panicle architecture variety from the perspective of population structure optimization but also provides a theoretical basis for supporting cultivation systems with the erect panicle architecture.

Multiple areas investigation reveals the genes related to vascular bundles in rice

BackgroundThe vascular bundle in the panicle neck is a crucial trait in rice (Oryza sativa) production that differs between the indica and japonica subspecies. However, the effect of indica/japonica genetic background on the vascular bundles remains unknown.ResultsA series of recombinant inbred lines (RILs) derived from a cross between japonica and indica were planted in three areas. High-throughput sequencing was conducted to determine the indica pedigree percentage and for quantitative trait locus (QTL) analysis. The indica pedigree affected the number of large vascular bundles (LVBs), but not the number of small vascular bundles (SVBs). QTL analysis identified a locus (qLVB9) that was pleiotropic for both LVBs and SVBs in all three areas, and qLVB9 appeared synonymous with DENSE AND ERECT PANICLE 1 (DEP1). Using CRISPR/Cas9 gene editing and gene overexpression technology, we confirmed that the truncated dep1 allele increased the number of LVBs, and resulted in LVBs more closely associated to the indica pedigree. RNA sequencing showed that the truncated dep1 allele downregulated the AP2-like gene family. The double mutant for the DEP1 and AP2-like genes (OsAP2–39) showed decreased endogenous abscisic acid (ABA) level and insensitivity to exogenous ABA treatment, confirming that both DEP1 and OsAP2–39 are involved in the ABA response mechanism.ConclusionsThe present study showed the qLVB9/DEP1 affects LVBs, and involved in ABA signaling via regulating the AP2-like gene family. These results offer new insights into the function of qLVB9/DEP1 in rice.

DEP1 is involved in regulating the carbon-nitrogen metabolic balance to affect grain yield and quality in rice (Oriza sativa L.).

The DEP1 (dense and erect panicle 1) gene, which corresponds to the erect panicle architecture, shows a pleiotropic effect in increasing grain yield and nitrogen use efficiency (NUE) in rice. Nevertheless, it remains unclear whether the carbon−nitrogen metabolic balance changes as the dep1 allele enhances nitrogen uptake and assimilation. In this study, we generated transgenic Akitakomati plants by overexpressing dep1 and analyzed the carbon−nitrogen metabolic status, gene expression profiles, and grain yield and quality. Under either low or high nitrogen growth conditions, the carbon−nitrogen metabolic balance of dep1-overexpressed lines was broken in stem sheaths and leaves but not in grains; the dep1-overexpressed plants showed higher expressions of glutamine synthetase (GS) and glutamate synthase (GOGAT) genes than the wildtype, along with increased total nitrogen and soluble protein content in the straw at maturity. However, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase (PEPC) genes were downregulated in dep1-overexpressed plants, leading to a decreased carbohydrate content and carbon/nitrogen ratio. Although the unbalanced carbon−nitrogen metabolism decreased the grain-filling rate, grain setting percentage, 1000 grain weight, and grain quality in dep1-overexpressed lines, it led to increased grain numbers per panicle and consequently increased grain yield. Our results suggest that an unbalanced carbon−nitrogen metabolic status is a major limiting factor for further improving grain yield and quality in erect panicle varieties.

Developing superior alleles of yield genes in rice by artificial mutagenesis using the CRISPR/Cas9 system

Rice yield is an important and complex agronomic trait controlled by multiple genes. In recent decades, dozens of yield-associated genes in rice have been cloned, many of which can increase production in the form of loss or degeneration of function. However, mutations occurring randomly under natural conditions have provided very limited genetic resources for yield increases. In this study, potentially yield-increasing alleles of two genes closely associated with yield were edited artificially. The recently developed CRISPR/Cas9 system was used to edit two yield genes: Grain number 1a (Gn1a) and DENSE AND ERECT PANICLE1 (DEP1). Several mutants were identified by a target sequence analysis. Phenotypic analysis confirmed one mutant allele of Gn1a and three of DEP1 conferring yield superior to that conferred by other natural high-yield alleles. Our results demonstrate that favorable alleles of the Gnla and DEP1 genes, which are considered key factors in rice yield increases, could be developed by artificial mutagenesis using genome editing technology.

Research progess of rice erect panicle gene <italic>DEP1</italic>

With the development of molecular biology, several genes relative to grain yield traits have been cloned in rice. Among these genes, only DEP1 (dense and erect panicle 1) has been widely used in rice breeding. The objective of this review is to summarize the previous research for DEP1 and look into its development prospect. DEP1 gene codes a heterotrimeric G protein γ subunit. The loss of its cysteine-rich domain increases signal transmission of G protein, which may result in stronger meristem activity, increased number of branches and grains per panicle, higher nitrogen use efficiency, and consequent increase in grain yield. Moreover, the erect panicle architectures improve the canopy structure and lodging resistance, which lead to higher material production capacity in population. Therefore, the plant type improvement on the basis of the erect panicle gene DEP1 will bring the third breakthrough of grain yield in rice breeding history after the semi-dwarf breeding and the application of heterosis in hybrid rice.

Deletion of a target gene in Indica rice via CRISPR/Cas9.

Using CRISPR/Cas9, we successfully deleted large fragments of the yield-related gene DENSE AND ERECT PANICLE1 in Indica rice at relatively high frequency and generated gain-of-function dep1 mutants. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a rapidly developing technology used to produce gene-specific modifications in both mammalian and plant systems. Most CRISPR-induced modifications in plants reported to date have been small insertions or deletions. Few large target gene deletions have thus far been reported, especially for Indica rice. In this study, we designed multiple CRISPR sgRNAs and successfully deleted DNA fragments in the gene DENSE AND ERECT PANICLE1 (DEP1) in the elite Indica rice line IR58025B. We achieved deletion frequencies of up to 21% for a 430bp target and 9% for a 10kb target among T0 events. Constructs with four sgRNAs did not generate higher full-length deletion frequencies than constructs with two sgRNAs. The multiple mutagenesis frequency reached 93% for four targets, and the homozygous mutation frequency reached 21% at the T0 stage. Important yield-related trait characteristics, such as dense and erect panicles and reduced plant height, were observed in dep1 homozygous T0 mutant plants produced by CRISPR/Cas9. Therefore, we successfully obtained deletions in DEP1 in the Indica background using the CRISPR/Cas9 editing tool at relatively high frequency.

Heterotrimeric G protein signaling in plant

Heterotrimeric guanine nucleotide-binding protein (G protein) signaling is an evolutionarily conserved mechanism in diverse eukaryotic organisms. In the past half century, five Nobel Prizes are awarded to researchers working in the G protein field, these seminal studies make the G protein pathway become the best understood signaling transmit system in the world. In animals, heterotrimeric G proteins contains α , β and γ subunits, perceives extracellular stimuli through G protein-coupled receptors (GPCR), and transmit signals to ion channels, enzymes and other effectors to affect numerous cellular behaviors. In recent years, much has been learned about the diversity of signal transduction through plant G proteins thanks to the identification and mutation of genes in Arabidopsis and rice. Plant cells have most of the core elements found in animal G signaling, such as α , β and γ subunits. However, plant G proteins transmit signals by atypical mechanisms. The plant G α subunit spontaneously releases GDP and forms a stable GTP bound state in vitro , and thus are self-activating. The self-activating characteristic also means that plant G protein paradigm do not need and therefore do not have typical animal GPCR. Indeed, the current evidence supports that no typical GPCR exists in plant. In most plants, the role of GPCR is performed by GTPase-accelerating protein (GAP), such as Arabidopsis regulator of G protein signaling 1 (AtRGS1). Interestingly, there is no ortholog of AtRGS1 in rice genome. Recently, an important advance has been proved that up-regulation of COLD1 is positively correlated with chilling tolerance in rice. COLD1 has nine-transmembrane domains, and localize to plasma membrane (PM) and endoplasmic reticulum (ER). The COLD1 physically interacts with RGA1 and acts as RGS protein that senses low temperature signals and accelerates G-protein GTPase activity. These results indicate that non-GPCR proteins in plant can topologically and functionally resemble animal GPCRs. In plants, the repertoire of the heterotrimeric G protein is much simpler than that in mammalian, but G γ subunits exhibit an extraordinary level of structural diversity and show significant differences from their animal counterparts. The non-canonical G γ subunits in plant kingdom have a large cysteine domain on C terminal, can be four times larger than the average mammalian G γ subunits size. The non-canonical G γ subunits present an inhibitory effects of N terminal on C terminal. Recent studies have shown that the manipulation of two non-canonical G γ subunits, GS3 (grain size 3) and DEP1 (dense and erect panicle 1), represents new strategy to simultaneously increase grain yield and nitrogen use efficiency in rice breeding. In this review, we focus on the recent insights of G protein network derived from the research about the model plant rice and Arabidopsis , the difference of G protein signal between plants and animals, and the potential of G protein to improve the crops productivity.

The DENSE AND ERECT PANICLE 1 (DEP1) gene offering the potential in the breeding of high-yielding rice.

The erect panicle model super-rice can rationally transform the solar energy into accumulated organic matter (biomass) and increase grain yield. The phenotype of erect panicle architecture controlled by DENSE AND ERECT PANICLE 1 (DEP1) has been used in rice breeding for nearly a century owing to its high-yield, lodging tolerance with strong stem, reasonable population structure and high nitrogen use efficiency. DEP1 is a G protein γ subunit that is involved in the regulation of erect panicle, number of grains per panicle, nitrogen uptake, and stress-tolerance through the G protein signal pathway. Here we review the development of erect panicle rice varieties, DEP1 alleles and regulatory network, and its physiological and morphological functions. Additionally, the further increasing the yield potential of erect-panicle super-rice, and the development of molecular designing breeding for indica-japonica hybrid rice with the dep1 gene are also prospected.

Variations in DENSE AND ERECT PANICLE 1 (DEP1) contribute to the diversity of the panicle trait in high-yielding japonica rice varieties in northern China.

Rice is one of mankind’s major food staples, and the erect panicle architecture in rice is an important morphological improvement. The dense and erect panicle 1 (DEP1) locus corresponds with the formation of erect panicles and has been widely used in rice breeding. However, the genetic diversity of DEP1 remains narrow. In order to improve the genetic diversity of DEP1, we used a rice germplasm collection of 72 high yielding japonica rice varieties to analyze the contribution of DEP1 to the panicle traits. We found 45 SNPs and 26 insertions and deletions (indels) within the DNA fragment of DEP1. We further detected 7 haplotypes and found that the replacement of 637 bp by a 12 bp fragment could explain the erect panicle architecture in all 72 germplasms. An SNP (G/C) at the -1253 bp of the promoter region caused a core sequence shift (TGGGCC) of a site II transcriptional regulatory element. The association analysis showed that the SNP(G/C) largely affects the number of primary and secondary branches, and grain number per panicle. Our results provide novel insights into the function and genetic diversity of DEP1. The SNP (G/C) at the promoter region will contribute to the flexible application of DEP1 in rice breeding.

Digital gene expression analysis reveals nitrogen fertilizer increases panicle size by repressing Hd3a signaling in rice

Nitrogen fertilizer significantly increases spikelet number and grain yield in rice. Although many transcriptome analyses of rice have been conducted, this is the first study to examine differential gene expression in young panicles of rice in response to nitrogen fertilizer. Digital gene expression tag profiling is a revolutionary approach for gene expression analysis. Here, differential gene expression was analyzed in young panicles that received different applications of fertilizer. Digital gene expression analysis identified hundreds of genes that significantly differed in their expression level after the plants received nitrogen treatment. Of note, nitrogen fertilizer significantly reduced several major photoperiod sensitivity regulators that trigger flowering in rice. Quantitative reverse transcription polymerase chain reaction analysis also showed that the expression of two florigen genes, Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1), was repressed in leaves by nitrogen fertilizer. In addition, the expression of two important genes that control panicle size, OsEATB and DENSE AND ERECT PANICLE1 (DEP1), was significantly promoted by nitrogen fertilizer. The identification of novel differentially expressed genes provides new insights into the profound impacts of nitrogen fertilizer on panicle development and may suggest new strategies to improve yield in rice.

Different effects of DEP1 on vascular bundle- and panicle-related traits under indica and japonica genetic backgrounds

Rice (Oryza sativa L.) is a major food for more than half of the world’s population and serves as a model monocot plant. DEP1 (dense and erect panicle 1), corresponding to an erect-panicle architecture, may bring the third breakthrough in rice breeding after the introduction of the semi-dwarf trait and the application of heterosis in hybrid rice. The aim of this study was to determine the effects of DEP1 on yield components and vascular bundle-related traits under indica and japonica genetic backgrounds. We analyzed a series of recombinant inbred lines, which were derived from a cross between the japonica variety SN265 with an erect panicle and the indica variety R99 with a curved panicle. The results showed that effects of the DEP1/dep1 allele were much stronger than the effects of the indica/japonica genetic background on vascular bundle-related traits and yield components. As the frequency of indica alleles increased, the grain yield per plant and the harvest index significantly increased in lines with the dep1 allele. Among the indica-type lines, those with the dep1 allele showed superior values for almost all of the vascular bundle-related traits and yield components. Our results provide new insights into the use of a high-yield-related gene derived from japonica in the indica genetic background.

A loss-of-function mutation of rice DENSE PANICLE 1 causes semi-dwarfness and slightly increased number of spikelets

In cereal breeding, semi-dwarfness and an increased spikelet number are favorable characteristics. We show that the rice DENSE PANICLE 1 (DN1) mutant allele Dn1-1 causes both of these characteristics and that Dn1-1 is a loss-of-function mutation. DN1 is allelic to DENSE AND ERECT PANICLE 1 (DEP1) (=qPE9-1). The expression level of OsCKX2 in the shoot apex of Dn1-1 plants is similar to that in the wild type, indicating that OsCKX2 does not contribute to an increased number of spikelets. A comparison of the Dn1-1 and Dn1-3 alleles suggests that the N-terminal region of DN1 contains a coiled-coil domain and a nuclear localization signal that might be responsible for semi-dwarfness. This comparison also revealed that a single transmembrane alpha-helix, a VWFC module, and a four-disulfide core domain can further increase spikelet number. Subcellular localization analysis of the DN1 protein fused with green fluorescent protein (GFP) implies that DN1 is located in the nucleus and cell membrane and that its N-terminal fragment is cleaved. Dn1-1 plants have normal sensitivity to gibberellin, brassinolide, and kinetin, and we observed no genetic epistasis with brassinolide-related mutants, suggesting that DN1 does not function in the signaling pathways of these phytohormones.