Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants.

Gravity controls directional growth of plants, and the classical starch-statolith hypothesis proposed more than a century ago postulates that amyloplast sedimentation in specialized cells initiates gravity sensing, but the molecular mechanism remains uncharacterized. The LAZY proteins are known as key regulators of gravitropism, and lazy mutants show striking gravitropic defects. Here, we report that gravistimulation by reorientation triggers mitogen-activated protein kinase (MAPK) signaling-mediated phosphorylation of Arabidopsis LAZY proteins basally polarized in root columella cells. Phosphorylation of LAZY increases its interaction with several translocons at the outer envelope membrane of chloroplasts (TOC) proteins on the surface of amyloplasts, facilitating enrichment of LAZY proteins on amyloplasts. Amyloplast sedimentation subsequently guides LAZY to relocate to the new lower side of the plasma membrane in columella cells, where LAZY induces asymmetrical auxin distribution and root differential growth. Together, this study provides a molecular interpretation for the starch-statolith hypothesis: the organelle-movement-triggered molecular polarity formation.

Analysis of the Complete Mitochondrial Genome of the Bitter Gourd (Momordica charantia).

Bitter gourd (Momordica charantia L.) is a significant vegetable. Although it has a special bitter taste, it is still popular with the public. The industrialization of bitter gourd could be hampered by a lack of genetic resources. The bitter gourd's mitochondrial and chloroplast genomes have not been extensively studied. In the present study, the mitochondrial genome of bitter gourd was sequenced and assembled, and its substructure was investigated. The mitochondrial genome of bitter gourd is 331,440 bp with 24 unique core genes, 16 variable genes, 3 rRNAs, and 23 tRNAs. We identified 134 SSRs and 15 tandem repeats in the entire mitochondrial genome of bitter gourd. Moreover, 402 pairs of repeats with a length greater than or equal to 30 were observed in total. The longest palindromic repeat was 523 bp, and the longest forward repeat was 342 bp. We found 20 homologous DNA fragments in bitter gourd, and the summary insert length was 19,427 bp, accounting for 5.86% of the mitochondrial genome. We predicted a total of 447 potential RNA editing sites in 39 unique PCGs and also discovered that the ccmFN gene has been edited the most often, at 38 times. This study provides a basis for a better understanding and analysis of differences in the evolution and inheritance patterns of cucurbit mitochondrial genomes.

Construction of a density mutant collection in bitter gourd via new germplasms innovation and gene functional study.

Although a few studies have elucidated the creation of bitter gourd mutants, the suitable concentration and duration of ethyl methanesulfonate (EMS) mutagenesis have not been determined. In this study, mutant collection was conducted to create new germplasms and widen genetic diversity. By employing the seeds of the inbred line Y52 as the mutagenic material, EMS as the mutagen, and the suitable mutagenic conditions for bitter gourd seeds (EMS concentration 0.2%, mutagenic time 10 h), we mutated 10,000 seeds and acquired 3223 independent M1 lines. For the randomly selected 1000 M2 lines, 199 M2 lines with visible phenotypes were found, and 167 M2 lines were mutants of fruit shape, size, and tubercles. Furthermore, fourteen dwarf, eleven leaf color, five leaf shape, and eight meristem defect mutants were discovered in this mutant collection. In addition, three lines of 1253, 2284, and 3269 represented recessive mutants crossed with Y52. Furthermore, the yellow leaf lines of 2284 and 3269 were not mutated at the same gene locus. This study constructed a mutant collection through innovative new germplasms and provided valuable resources for bitter gourd breeding and functional gene research.

SAUR17 and SAUR50 Differentially Regulate PP2C-D1 during Apical Hook Development and Cotyledon Opening in Arabidopsis.

Following germination in the dark, Arabidopsis (Arabidopsis thaliana) seedlings undergo etiolation and develop apical hooks, closed cotyledons, and rapidly elongating hypocotyls. Upon light perception, the seedlings de-etiolate, which includes the opening of apical hooks and cotyledons. Here, we identify Arabidopsis Small Auxin Up RNA17 (SAUR17) as a downstream effector of etiolation, which serves to bring about apical hook formation and closed cotyledons. SAUR17 is highly expressed in apical hooks and cotyledons and is repressed by light. The apical organs also express a group of light-inducing SAURs, as represented by SAUR50, which promote hook and cotyledon opening. The development of etiolated or de-etiolated apical structures requires asymmetric differential cell growth. We present evidence that the opposing actions of SAUR17 and SAUR50 on apical development largely result from their antagonistic regulation of Protein Phosphatase 2C D-clade 1 (PP2C-D1), a phosphatase that suppresses cell expansion and promotes apical hook development in the dark. SAUR50 inhibits PP2C-D1, whereas SAUR17 has a higher affinity for PP2C-D1 without inhibiting its activity. PP2C-D1 predominantly associates with SAUR17 in etiolated seedlings, which shields it from inhibitory SAURs such as SAUR50. Light signals turn off SAUR17 and upregulate a subgroup of SAURs including SAUR50 at the inner side of the hook and cotyledon cells, leading to cell expansion and unfolding of the hook and cotyledons.

Light modulates the gravitropic responses through organ-specific PIFs and HY5 regulation of LAZY4 expression in Arabidopsis.

Light and gravity are two key environmental factors that control plant growth and architecture. However, the molecular basis of the coordination of light and gravity signaling in plants remains obscure. Here, we report that two classes of transcription factors, PHYTOCHROME INTERACTING FACTORS (PIFs) and ELONGATED HYPOCOTYL5 (HY5), can directly bind and activate the expression of LAZY4, a positive regulator of gravitropism in both shoots and roots in Arabidopsis In hypocotyls, light promotes degradation of PIFs to reduce LAZY4 expression, which inhibits the negative gravitropism of hypocotyls. LAZY4 overexpression can partially rescue the negative gravitropic phenotype of pifq in the dark without affecting amyloplast development. Our identification of the PIFs-LAZY4 regulatory module suggests the presence of another role for PIF proteins in gravitropism, in addition to a previous report demonstrating that PIFs positively regulate amyloplast development to promote negative gravitropism in hypocotyls. In roots, light promotes accumulation of HY5 proteins to activate expression of LAZY4, which promotes positive gravitropism in roots. Together, our data indicate that light exerts opposite regulation of LAZY4 expression in shoots and roots by mediating the protein levels of PIFs and HY5, respectively, to inhibit the negative gravitropism of shoots and promote positive gravitropism of roots in Arabidopsis.

The Asymmetric Expression of SAUR Genes Mediated by ARF7/19 Promotes the Gravitropism and Phototropism of Plant Hypocotyls.

The asymmetric distribution of auxin leads to the bending growth of hypocotyls during gravitropic and phototropic responses, but the signaling events downstream of auxin remain unclear. Here, we identify many SAUR genes showing asymmetric expression in soybean hypocotyls during gravistimulation and then study their homologs in Arabidopsis. SAUR19 subfamily genes have asymmetric expression in Arabidopsis hypocotyls during gravitropic and phototropic responses, induced by the lateral redistribution of auxin. Both the mutation of SAUR19 subfamily genes and the ectopic expression of SAUR19 weaken these tropic responses, indicating the critical role of their asymmetric expression. The auxin-responsive transcription factor ARF7 may directly bind the SAUR19 promoter and activate SAUR19 expression asymmetrically in tropic responses. Taken together, our results reveal that a gravity- or light-triggered asymmetric auxin distribution induces the asymmetric expression of SAUR19 subfamily genes by ARF7 and ARF19 in the hypocotyls, which leads to bending growth during gravitropic and phototropic responses.

Using Genetic Engineering Techniques to Develop Banana Cultivars With Fusarium Wilt Resistance and Ideal Plant Architecture.

Bananas (Musa spp.) are an important fruit crop worldwide. The fungus Fusarium oxysporum f. sp. cubense (Foc), which causes Fusarium wilt, is widely regarded as one of the most damaging plant diseases. Fusarium wilt has previously devastated global banana production and continues to do so today. In addition, due to the current use of high-density banana plantations, desirable banana varieties with ideal plant architecture (IPA) possess high lodging resistance, optimum photosynthesis, and efficient water absorption. These properties may help to increase banana production. Genetic engineering is useful for the development of banana varieties with Foc resistance and ideal plant architecture due to the sterility of most cultivars. However, the sustained immune response brought about by genetic engineering is always accompanied by yield reductions. To resolve this problem, we should perform functional genetic studies of the Musa genome, in conjunction with genome editing experiments, to unravel the molecular mechanisms underlying the immune response and the formation of plant architecture in the banana. Further explorations of the genes associated with Foc resistance and ideal architecture might lead to the development of banana varieties with both ideal architecture and pathogen super-resistance. Such varieties will help the banana to remain a staple food worldwide.

Improved de novo genome assembly and analysis of the Chinese cucurbit Siraitia grosvenorii, also known as monk fruit or luo-han-guo.

Background: Luo-han-guo (Siraitia grosvenorii), also called monk fruit, is a member of the Cucurbitaceae family. Monk fruit has become an important area for research because of the pharmacological and economic potential of its noncaloric, extremely sweet components (mogrosides). It is also commonly used in traditional Chinese medicine for the treatment of lung congestion, sore throat, and constipation. Recently, a single reference genome became available for monk fruit, assembled from 36.9x genome coverage reads via Illumina sequencing platforms. This genome assembly has a relatively short (34.2 kb) contig N50 length and lacks integrated annotations. These drawbacks make it difficult to use as a reference in assembling transcriptomes and discovering novel functional genes. Findings: Here, we offer a new high-quality draft of the S. grosvenorii genome assembled using 31 Gb (∼73.8x) long single molecule real time sequencing reads and polished with ∼50 Gb Illumina paired-end reads. The final genome assembly is approximately 469.5 Mb, with a contig N50 length of 432,384 bp, representing a 12.6-fold improvement. We further annotated 237.3 Mb of repetitive sequence and 30,565 consensus protein coding genes with combined evidence. Phylogenetic analysis showed that S. grosvenorii diverged from members of the Cucurbitaceae family approximately 40.9 million years ago. With comprehensive transcriptomic analysis and differential expression testing, we identified 4,606 up-regulated genes in the early fruit compared to the leaf, a number of which were linked to metabolic pathways regulating fruit development and ripening. Conclusions: The availability of this new monk fruit genome assembly, as well as the annotations, will facilitate the discovery of new functional genes and the genetic improvement of monk fruit.

Light-Dependent Degradation of PIF3 by SCFEBF1/2 Promotes a Photomorphogenic Response in Arabidopsis.

Plant seedlings emerging from darkness into the light environment undergo photomorphogenesis, which enables autotrophic growth with optimized morphology and physiology. During this transition, plants must rapidly remove photomorphogenic repressors accumulated in the dark. Among them is PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), a key transcription factor promoting hypocotyl growth. Here we report that, in response to light activation of phytochrome photoreceptors, EIN3-BINDING F BOX PROTEINs (EBFs) 1 and 2 mediate PIF3 protein degradation in a manner dependent on light-induced phosphorylation of PIF3. Whereas PIF3 binds EBFs independent of light, the recruitment of PIF3-EBFs to the core SKP1-CUL1-F box protein (SCF) scaffold is facilitated by light signals or PIF3 phosphorylation. We also found that previously identified LIGHT-RESPONSE BRIC-A-BRACK/TRAMTRACK/BROAD (LRB) E3 ubiquitin ligases target phytochrome B (phyB) and PIF3 primarily under high-light conditions, whereas EBF1/2 vigorously target PIF3 degradation under wide ranges of light intensity without affecting the abundance of phyB. Both genetic and molecular data support that SCFEBF1/2 function as photomorphogenic E3s during seedling development.

Genome-wide study of an elite rice pedigree reveals a complex history of genetic architecture for breeding improvement.

Improving breeding has been widely utilized in crop breeding and contributed to yield and quality improvement, yet few researches have been done to analyze genetic architecture underlying breeding improvement comprehensively. Here, we collected genotype and phenotype data of 99 cultivars from the complete pedigree including Huanghuazhan, an elite, high-quality, conventional indica rice that has been grown over 4.5 million hectares in southern China and from which more than 20 excellent cultivars have been derived. We identified 1,313 selective sweeps (SSWs) revealing four stage-specific selection patterns corresponding to improvement preference during 65 years, and 1113 conserved Huanghuazhan traceable blocks (cHTBs) introduced from different donors and conserved in >3 breeding generations were the core genomic regions for superior performance of Huanghuazhan. Based on 151 quantitative trait loci (QTLs) identified for 13 improved traits in the pedigree, we reproduced their improvement process in silico, highlighting improving breeding works well for traits controlled by major/major + minor effect QTLs, but was inefficient for traits controlled by QTLs with complex interactions or explaining low levels of phenotypic variation. These results indicate long-term breeding improvement is efficient to construct superior genetic architecture for elite performance, yet molecular breeding with designed genotype of QTLs can facilitate complex traits improvement.

DELLA-mediated PIF degradation contributes to coordination of light and gibberellin signalling in Arabidopsis.

Light and gibberellins (GAs) antagonistically regulate hypocotyl elongation in plants. It has been demonstrated that DELLAs, which are negative regulators of GA signalling, inhibit phytochrome-interacting factors 3 and 4 (PIF3 and PIF4) by sequestering their DNA-recognition domains. However, it is unclear whether there are other mechanisms of regulatory crosstalk between DELLAs and PIFs. Here, we demonstrate that DELLAs negatively regulate the abundance of four PIF proteins through the ubiquitin-proteasome system. Reduction of PIF3 protein abundance by DELLAs correlates closely with reduced hypocotyl elongation. Both sequestration and degradation of PIF3 by DELLAs contribute to a reduction in PIF3 binding to its target genes. Thus, we show that promotion of PIF degradation by DELLAs is required to coordinate light and GA signals, and the dual regulation of transcription factors by DELLAs by both sequestration and degradation may be a general mechanism.

Exploring the genetic characteristics of 93-11 and Nipponbare recombination inbred lines based on the GoldenGate SNP assay.

Understanding genetic characteristics in rice populations will facilitate exploring evolutionary mechanisms and gene cloning. Numerous molecular markers have been utilized in linkage map construction and quantitative trait locus (QTL) mappings. However, segregation-distorted markers were rarely considered, which prevented understanding genetic characteristics in many populations. In this study, we designed a 384-marker GoldenGate SNP array to genotype 283 recombination inbred lines (RILs) derived from 93-11 and Nipponbare Oryza sativa crosses. Using 294 markers that were highly polymorphic between parents, a linkage map with a total genetic distance of 1,583.2 cM was constructed, including 231 segregation-distorted markers. This linkage map was consistent with maps generated by other methods in previous studies. In total, 85 significant quantitative trait loci (QTLs) with phenotypic variation explained (PVE) values≥5% were identified. Among them, 34 QTLs were overlapped with reported genes/QTLs relevant to corresponding traits, and 17 QTLs were overlapped with reported sterility-related genes/QTLs. Our study provides evidence that segregation-distorted markers can be used in linkage map construction and QTL mapping. Moreover, genetic information resulting from this study will help us to understand recombination events and segregation distortion. Furthermore, this study will facilitate gene cloning and understanding mechanism of inter-subspecies hybrid sterility and correlations with important agronomic traits in rice.

Pedigree-based analysis of derivation of genome segments of an elite rice reveals key regions during its breeding.

Analyses of genome variations with high-throughput assays have improved our understanding of genetic basis of crop domestication and identified the selected genome regions, but little is known about that of modern breeding, which has limited the usefulness of massive elite cultivars in further breeding. Here we deploy pedigree-based analysis of an elite rice, Huanghuazhan, to exploit key genome regions during its breeding. The cultivars in the pedigree were resequenced with 7.6× depth on average, and 2.1 million high-quality single nucleotide polymorphisms (SNPs) were obtained. Tracing the derivation of genome blocks with pedigree and information on SNPs revealed the chromosomal recombination during breeding, which showed that 26.22% of Huanghuazhan genome are strictly conserved key regions. These major effect regions were further supported by a QTL mapping of 260 recombinant inbred lines derived from the cross of Huanghuazhan and a very dissimilar cultivar, Shuanggui 36, and by the genome profile of eight cultivars and 36 elite lines derived from Huanghuazhan. Hitting these regions with the cloned genes revealed they include numbers of key genes, which were then applied to demonstrate how Huanghuazhan were bred after 30 years of effort and to dissect the deficiency of artificial selection. We concluded the regions are helpful to the further breeding based on this pedigree and performing breeding by design. Our study provides genetic dissection of modern rice breeding and sheds new light on how to perform genomewide breeding by design.

De novo assembly and comparative analysis of root transcriptomes from different varieties of Panax ginseng C. A. Meyer grown in different environments.

Panax ginseng C. A. Meyer is an important traditional herb in eastern Asia. It contains ginsenosides, which are primary bioactive compounds with medicinal properties. Although ginseng has been cultivated since at least the Ming dynasty to increase production, cultivated ginseng has lower quantities of ginsenosides and lower disease resistance than ginseng grown under natural conditions. We extracted root RNA from six varieties of fifth-year P. ginseng cultivars representing four different growth conditions, and performed Illumina paired-end sequencing. In total, 163,165,706 raw reads were obtained and used to generate a de novo transcriptome that consisted of 151,763 contigs (76,336 unigenes), of which 100,648 contigs (66.3%) were successfully annotated. Differential expression analysis revealed that most differentially expressed genes (DEGs) were upregulated (246 out of 258, 95.3%) in ginseng grown under natural conditions compared with that grown under artificial conditions. These DEGs were enriched in gene ontology (GO) terms including response to stimuli and localization. In particular, some key ginsenoside biosynthesis-related genes, including HMG-CoA synthase (HMGS), mevalonate kinase (MVK), and squalene epoxidase (SE), were upregulated in wild-grown ginseng. Moreover, a high proportion of disease resistance-related genes were upregulated in wild-grown ginseng. This study is the first transcriptome analysis to compare wild-grown and cultivated ginseng, and identifies genes that may produce higher ginsenoside content and better disease resistance in the wild; these genes may have the potential to improve cultivated ginseng grown in artificial environments.

Arabidopsis DE-ETIOLATED1 represses photomorphogenesis by positively regulating phytochrome-interacting factors in the dark.

Arabidopsis thaliana seedlings undergo photomorphogenic development even in darkness when the function of DE-ETIOLATED1 (DET1), a repressor of photomorphogenesis, is disrupted. However, the mechanism by which DET1 represses photomorphogenesis remains unclear. Our results indicate that DET1 directly interacts with a group of transcription factors known as the phytochrome-interacting factors (PIFs). Furthermore, our results suggest that DET1 positively regulates PIF protein levels primarily by stabilizing PIF proteins in the dark. Genetic analysis showed that each pif single mutant could enhance the det1-1 phenotype, and ectopic expression of each PIF in det1-1 partially suppressed the det1-1 phenotype, based on hypocotyl elongation and cotyledon opening angles observed in darkness. Genomic analysis also revealed that DET1 may modulate the expression of light-regulated genes to mediate photomorphogenesis partially through PIFs. The observed interaction and regulation between DET1 and PIFs not only reveal how DET1 represses photomorphogenesis, but also suggest a possible mechanism by which two groups of photomorphogenic repressors, CONSTITUTIVE PHOTOMORPHOGENESIS/DET/FUSCA and PIFs, work in concert to repress photomorphogenesis in darkness.

A high-density SNP genotyping array for rice biology and molecular breeding.

A high-density single nucleotide polymorphism (SNP) array is critically important for geneticists and molecular breeders. With the accumulation of huge amounts of genomic re-sequencing data and available technologies for accurate SNP detection, it is possible to design high-density and high-quality rice SNP arrays. Here we report the development of a high-density rice SNP array and its utility. SNP probes were designed by screening more than 10 000 000 SNP loci extracted from the re-sequencing data of 801 rice varieties and an array named RiceSNP50 was produced on the Illumina Infinium platform. The array contained 51 478 evenly distributed markers, 68% of which were within genic regions. Several hundred rice plants with parent/F1 relationships were used to generate a high-quality cluster file for accurate SNP calling. Application tests showed that this array had high genotyping accuracy, and could be used for different objectives. For example, a core collection of elite rice varieties was clustered with fine resolution. Genome-wide association studies (GWAS) analysis correctly identified a characterized QTL. Further, this array was successfully used for variety verification and trait introgression. As an accurate high-throughput genotyping tool, RiceSNP50 will play an important role in both functional genomics studies and molecular breeding.

Highly efficient genotyping of rice biparental populations by GoldenGate assays based on parental resequencing.

KEY MESSAGE: A new time- and cost-effective strategy was developed for medium-density SNP genotyping of rice biparental populations, using GoldenGate assays based on parental resequencing. Since the advent of molecular markers, crop researchers and breeders have dedicated huge amounts of effort to detecting quantitative trait loci (QTL) in biparental populations for genetic analysis and marker-assisted selection (MAS). In this study, we developed a new time- and cost-effective strategy for genotyping a population of progeny from a rice cross using medium-density single nucleotide polymorphisms (SNPs). Using this strategy, 728,362 "high quality" SNPs were identified by resequencing Teqing and Lemont, the parents of the population. We selected 384 informative SNPs that were evenly distributed across the genome for genotyping the biparental population using the Illumina GoldenGate assay. 335 (87.2 %) validated SNPs were used for further genetic analyses. After removing segregation distortion markers, 321 SNPs were used for linkage map construction and QTL mapping. This strategy generated SNP markers distributed more evenly across the genome than previous SSR assays. Taking the GW5 gene that controls grain shape as an example, our strategy provided higher accuracy (0.8 Mb) and significance (LOD 5.5 and 10.1) in QTL mapping than SSR analysis. Our study thus provides a rapid and efficient strategy for genetic studies and QTL mapping using SNP genotyping assays.

Development and application of a set of breeder-friendly SNP markers for genetic analyses and molecular breeding of rice (Oryza sativa L.).

Single nucleotide polymorphisms (SNPs) are the most abundant DNA markers in plant genomes. In this study, based on 54,465 SNPs between the genomes of two Indica varieties, Minghui 63 (MH63) and Zhenshan 97 (ZS97) and additional 20,705 SNPs between the MH63 and Nipponbare genomes, we identified and confirmed 1,633 well-distributed SNPs by PCR and Sanger sequencing. From these, a set of 372 SNPs were further selected to analyze the patterns of genetic diversity in 300 representative rice inbred lines from 22 rice growing countries worldwide. Using this set of SNPs, we were able to uncover the well-known Indica-Japonica subspecific differentiation and geographic differentiations within Indica and Japonica. Furthermore, our SNP results revealed some common and contrasting patterns of the haplotype diversity along different rice chromosomes in the Indica and Japonica accessions, which suggest different evolutionary forces possibly acting in specific regions of the rice genome during domestication and evolution of rice. Our results demonstrated that this set of SNPs can be used as anchor SNPs for large scale genotyping in rice molecular breeding research involving Indica-Japonica and Indica-Indica crosses.