Integrated analysis of metabolome and transcriptome reveals key candidate genes involved in flavonoid biosynthesis in Pinellia ternata under heat stress.

Pinellia ternata (Thunb.) Breit. is an important traditional Chinese medicinal herb and very sensitive to high temperatures. To gain a better understanding of flavonoid biosynthesis under heat stress in P. ternata, we performed integrated analyses of metabolome and transcriptome data. P. ternata plants were subjected to a temperature of 38 °C, and samples were collected after 10 d of treatment. A total of 502 differential accumulated metabolites and 5040 different expressed transcripts were identified, with flavonoid biosynthesis predominantly enriched. Integrated metabolomics and transcriptome analysis showed that high temperature treatment upregulated the expression of CYP73A and downregulated the expression of other genes (such as HCT, CCoAOMT, DFR1, DFR2), which might inhibit the biosynthesis of the downstream metabolome, including such metabolites as chlorogenic acid, pelargonidin, cyanidin, and (-)-epigallocatechin in the flavonoid biosynthesis pathway. The transcription expression levels of these genes were validated by real-time PCR. Our results provide valuable insights into flavonoid composition and accumulation patterns and the candidate genes participating in the flavonoid biosynthesis pathways under heat stress in P. ternata.

The chromosome-level reference genome of Coptis chinensis provides insights into genomic evolution and berberine biosynthesis.

Coptis chinensis Franch, a perennial herb, is mainly distributed in southeastern China. The rhizome of C. chinensis has been used as a traditional medicine for more than 2000 years in China and many other Asian countries. The pharmacological activities of C. chinensis have been validated by research. Here, we present a de novo high-quality genome of C. chinensis with a chromosome-level genome of ~958.20 Mb, a contig N50 of 1.58 Mb, and a scaffold N50 of 4.53 Mb. We found that the relatively large genome size of C. chinensis was caused by the amplification of long terminal repeat (LTR) retrotransposons. In addition, a whole-genome duplication event in ancestral Ranunculales was discovered. Comparative genomic analysis revealed that the tyrosine decarboxylase (TYDC) and (S)-norcoclaurine synthase (NCS) genes were expanded and that the aspartate aminotransferase gene (ASP5) was positively selected in the berberine metabolic pathway. Expression level and HPLC analyses showed that the berberine content was highest in the roots of C. chinensis in the third and fourth years. The chromosome-level reference genome of C. chinensis provides important genomic data for molecular-assisted breeding and active ingredient biosynthesis.

Characterization of a novel allele of bc12/gdd1 indicates a differential leaf color function for BC12/GDD1 in Indica and Japonica backgrounds.

Leaf color is directly associated with plant photosynthesis. Here, we have isolated and identified a spontaneous rice mutant named yd1 that has yellowish leaves and dwarf stature. Map-based cloning reveals that YD1 encodes a previously reported kinesin protein from the kinesin-4 subfamily, BC12/GDD1. Arginine-328 is replaced by leucine in yd1, BC12328Leu. YD1 is mainly expressed in leaves and is involved in chlorophyll (Chl) synthesis. The yd1 mutant had less Chl and a reduced and disordered thylakoid ultrastructure. In yd1 plants, Chl biosynthesis and photosynthesis associated gene expression was decreased and Chl degradation gene expression was increased, thereby leading to a reduced photosynthesis rate and grain yield. In this study we reveal that the novel BC12328Leu allele of BC12 modulated plant leaf color in yd1 plants, which has not been previously reported in studies of BC12/GDD1/MTD1/SRG1. Gene knockout results indicated that YD1 regulates leaf color in the indica rice background, but not in the japonica rice background. Our study provides new insights into molecular regulation of rice growth by BC12/GDD1 in different genetic backgrounds.

GWC1 is essential for high grain quality in rice.

Appearance quality is an important determinant of rice quality. Many genes that affect grain appearance quality have been identified, but the regulatory mechanisms that contribute to this trait remain unclear. Here, two grains with chalkiness (gwc1) mutants, gwc1-1 and gwc1-2, were identified from an EMS-mutagenized population of indica rice cultivar Shuhui498 (R498). The gwc1 mutants had poor grain appearance quality consistent with the measured values for the percentage of grains with chalkiness, square of chalky endosperm, the total starch, amylose and sucrose contents. Milling quality and grain size were also affected in the gwc1 mutants. The gwc1-1 and gwc1-2 were found to be loss-of-function allelic mutants. GWC1 was mapped to the long arm of rice chromosome 8 using the MutMap strategy and incorrectly annotated in the reference genome for Nipponbare (MSU). The GWC1 gene corresponds to the WTG1/OsOTUB1 gene, which encodes an otubain-like protease with deubiquitinating activity that is homologous to human OTUB1. GWC1 transcripts accumulated to high levels in early endosperm after fertilization and developing inflorescences, and GWC1-green fluorescent protein (GFP) signal was detected in the nucleus and cytoplasm. GWC1 is likely to regulate grain appearance quality through genes involved in sucrose metabolism and starch biosynthesis. Overall, the present findings reveal that GWC1 is important for grain quality and yield due to its effects on grain chalkiness and size.

Mitochondria-Associated Pyruvate Kinase Complexes Regulate Grain Filling in Rice.

Grain filling is a complex agronomic trait that directly determines grain weight and quality in rice (Oryza sativa). Nevertheless, key factors affecting grain filling remain poorly understood. Here, we identified a grain filling gene, OsPK3, encoding a pyruvate kinase (PK). The loss of function of OsPK3 caused reduced PK activity and Suc translocation defects from source to sink in rice, which led to compromised grain filling. OsPK3 was constitutively expressed but had relatively higher expression levels in leaf and developing caryopsis and specific expression signals in tissues involved in Suc transport and unloading, supporting its biological function in regulation of grain filling by affecting Suc translocation. Subcellular localization analysis of OsPK3 revealed its association with mitochondria, and OsPK3 physically interacted and formed heterodimers in vivo with two other PK isozymes, OsPK1 and OsPK4. Both OsPK1 and OsPK4 localized to the mitochondria and cytosol and were recruited to the mitochondria by OsPK3. Despite their high sequence similarity, OsPK1 and OsPK4 had distinct expression patterns. As observed for ospk3, disruption of OsPK1 caused pleiotropic defects, while OsPK4 loss of function led to severely chalky grains without other obvious defects. Collectively, we revealed that two mitochondria-associated pyruvate kinase complexes, OsPK3-OsPK1/OsPK4, are involved in regulation of grain filling by stage-specific fine-tuning of Suc translocation.

[Genetic analysis of dense and erect panicle 2 allele DEP2-1388 and its application in hybrid rice breeding].

Using ethyl methanesulfonate (EMS) mutagenesis, we isolated an erect panicle mutant, R1338, from indica heavy-panicle restorer Shuhui498. Compared with wild type control, the mutant displayed dwarfism, erect and short panicle, short primary panicle branch, increased grain density, short grain length and increased grain thickness. In addition, the erect panicle architecture of R1388 resulted in significant decreased bending moment and increased resistance to panicle bending. Histocytological analysis indicated that the diameter of uppermost internode, cellulose content and lignin content play important roles in resistance to panicle bending. Genetic analysis revealed that the mutant phenotype was controlled by a semi-dominant nuclear gene. With resequencing and MutMap analysis strategy, we found that one SNP from A to G at the seventh exon of DEP2 resulted in the 928(th) amino acid substitution from arginine (AGG) to glycine (GGG) in R1338 mutant. Considering the phenotype of other dep2 mutants, the phenotype of R1338 was likely to be caused by the SNP in DEP2. The mutant R1338 and wild type were crossed with several sterile lines which respectively had different panicle types, the combinations generated from R1338 and curve panicle sterile lines showed semi-erect panicle, higher seed setting percentage and heterosis, and the combinations generated from R1388 and erect panicle sterile line with DEP1 showed erect panicle by gene additive effect. Moreover, the combinations with semi-erect panicle had superior light transmittance and stronger light intensity, which improved efficiency of light utilization to intermediate and subjacent leaves compared to the combinations with curved panicle. This study provides a good strategy to solve the problem of population density in three-line hybrid rice.