Hongmei Cai

Accumulated Expression Level of Cytosolic Glutamine Synthetase 1 Gene (OsGS1;1 or OsGS1;2) Alter Plant Development and the Carbon-Nitrogen Metabolic Status in Rice

Maintaining an appropriate balance of carbon to nitrogen metabolism is essential for rice growth and yield. Glutamine synthetase is a key enzyme for ammonium assimilation. In this study, we systematically analyzed the growth phenotype, carbon-nitrogen metabolic status and gene expression profiles in GS1;1-, GS1;2-overexpressing rice and wildtype plants. Our results revealed that the GS1;1-, GS1;2-overexpressing plants exhibited a poor plant growth phenotype and yield and decreased carbon/nitrogen ratio in the stem caused by the accumulation of nitrogen in the stem. In addition, the leaf SPAD value and photosynthetic parameters, soluble proteins and carbohydrates varied greatly in the GS1;1-, GS1;2-overexpressing plants. Furthermore, metabolite profile and gene expression analysis demonstrated significant changes in individual sugars, organic acids and free amino acids, and gene expression patterns in GS1;1-, GS1;2-overexpressing plants, which also indicated the distinct roles that these two GS1 genes played in rice nitrogen metabolism, particularly when sufficient nitrogen was applied in the environment. Thus, the unbalanced carbon-nitrogen metabolic status and poor ability of nitrogen transportation from stem to leaf in GS1;1-, GS1;2-overexpressing plants may explain the poor growth and yield.

Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status.

AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

The Stable Level of Glutamine synthetase 2 Plays an Important Role in Rice Growth and in Carbon-Nitrogen Metabolic Balance

Glutamine synthetase 2 (GS2) is a key enzyme involved in the ammonium metabolism in plant leaves. In our previous study, we obtained GS2-cosuppressed plants, which displayed a normal growth phenotype at the seedling stage, while at the tillering stage they showed a chlorosis phenotype. In this study, to investigate the chlorosis mechanism, we systematically analyzed the plant growth, carbon-nitrogen metabolism and gene expressions between the GS2-cosuppressed rice and wild-type plants. The results revealed that the GS2-cosuppressed plants exhibited a poor plant growth phenotype and a poor nitrogen transport ability, which led to nitrogen accumulation and a decline in the carbon/nitrogen ratio in the stems. Interestingly, there was a higher concentration of soluble proteins and a lower concentration of carbohydrates in the GS2-cosuppressed plants at the seedling stage, while a contrasting result was displayed at the tillering stage. The analysis of the metabolic profile showed a significant increase of sugars and organic acids. Additionally, gene expression patterns were different in root and leaf of GS2-cosuppressed plants between the seedling and tillering stage. These results indicated the important role of a stable level of GS2 transcription during normal rice development and the importance of the carbon-nitrogen metabolic balance in rice growth.

The boron transporter BnaC4.BOR1;1c is critical for inflorescence development and fertility under boron limitation in Brassica napus

Boron (B) is an essential micronutrient for plants, but the molecular mechanisms underlying the uptake and distribution of B in allotetraploid rapeseed (Brassica napus) are unclear. Here, we identified a B transporter of rapeseed, BnaC4.BOR1;1c, which is expressed in shoot nodes and involved in distributing B to the reproductive organs. Transgenic Arabidopsis plants containing a BnaC4.BOR1;1c promoter-driven GUS reporter gene showed strong GUS activity in roots, nodal regions of the shoots and immature floral buds. Overexpressing BnaC4.BOR1;1c in Arabidopsis wild type or in bor1-1 mutants promoted wild-type growth and rescued the bor1-1 mutant phenotype. Conversely, knockdown of BnaC4.BOR1;1c in a B-efficient rapeseed line reduced B accumulation in flower organs, eventually resulting in severe sterility and seed yield loss. BnaC4.BOR1;1c RNAi plants exhibited large amounts of disintegrated stigma papilla cells with thickened cell walls accompanied by abnormal proliferation of lignification under low-B conditions, indicating that the sterility may be a result of altered cell wall properties in flower organs. Taken together, our results demonstrate that BnaC4.BOR1;1c is a AtBOR1-homologous B transporter gene expressing in both roots and shoot nodes that is essential for the developing inflorescence tissues, which highlights its diverse functions in allotetraploid rapeseed compared with diploid model plant Arabidopsis.

Identification and characterization of improved nitrogen efficiency in interspecific hybridized new-type Brassica napus

BACKGROUND AND AIMS Oilseed rape (Brassica napus) is an important oil crop worldwide. The aim of this study was to identify the variation in nitrogen (N) efficiency of new-type B. napus (genome A(r)A(r)C(c)C(c)) genotypes, and to characterize some critical physiological and molecular mechanisms in response to N limitation. METHODS Two genotypes with contrasting N efficiency (D4-15 and D1-1) were identified from 150 new-type B. napus lines, and hydroponic and pot experiments were conducted. Root morphology, plant biomass, N uptake parameters and seed yield of D4-15 and D1-1 were investigated. Two traditional B. napus (genome A(n)A(n)C(n)C(n)) genotypes, QY10 and NY7, were also cultivated. Introgression of exotic genomic components in D4-15 and D1-1 was evaluated with molecular markers. KEY RESULTS Large genetic variation existed among traits contributing to the N efficiency of new-type B. napus. Under low N levels at the seedling stage, the N-efficient new-type D4-15 showed higher values than the N-inefficient D1-1 line and the traditional B. napus QY10 and NY7 genotypes with respect to several traits, including root and shoot biomass, root morphology, N accumulation, N utilization efficiency (NutE), N uptake efficiency (NupE), activities of nitrate reductase (NR) and glutamine synthetase (GS), and expression levels of N transporter genes and genes that are involved in N assimilation. Higher yield was produced by the N-efficient D4-15 line compared with the N-inefficient D1-1 at maturity. More exotic genome components were introgressed into the genome of D4-15 (64·97 %) compared with D1-1 (32·23 %). CONCLUSIONS The N-efficient new-type B. napus identified in this research had higher N efficiency (and tolerance to low-N stress) than traditional B. napus cultivars, and thus could have important potential for use in breeding N-efficient B. napus cultivars in the field.

Characterization of phosphorus starvation-induced gene BnSPX3 in Brassica napus

Background and aimsThe molecular studies of Pi starvation in Brassica napus have been limited. To explore the molecular responses and mechanisms involved in Pi starvation in Brassica napus, two SPX domain genes, BnSPX3;1 and BnSPX3;2, were isolated.MethodsSolution culture, DNA and RNA isolation, gene cloning, RT-PCR, construction of vector and transformation, GUS staining and bioinformatic analysis were used.ResultsBnSPX3;1 and BnSPX3;2 are 936 and 863 bp in length and have similar structures with three exons and two introns. Their expression was significantly induced by Pi starvation but not induced by other nutrients (N, K, S, Fe) starvation or salt stress, osmotic stress, heavy metal toxicity. The induction was rapid and durative during Pi starvation, and was reversible upon resupply of Pi. The expression level of both genes was dependent on external Pi concentration and decreased with increasing of Pi concentration. Furthermore, their promoters were isolated, each of which contains four P1BS elements. Both promoters were fused to GUS and transformed into Arabidopsis, respectively. The GUS activity was only observed in P-starved plants but not in plants starved with other nutrients (N, K, S, Fe).ConclusionsThese results suggest that both BnSPX3;1 and BnSPX3;2 are P-specific genes in Brassica napus, and their expression level can be used as biomarker to evaluate plant Pi-starvation status and their promoters can be used as P-specific inducible promoter in nutrient genetic engineering.

A Natural Light/Dark Cycle Regulation of Carbon-Nitrogen Metabolism and Gene Expression in Rice Shoots.

Light and temperature are two particularly important environmental cues for plant survival. Carbon and nitrogen are two essential macronutrients required for plant growth and development, and cellular carbon and nitrogen metabolism must be tightly coordinated. In order to understand how the natural light/dark cycle regulates carbon and nitrogen metabolism in rice plants, we analyzed the photosynthesis, key carbon-nitrogen metabolites, and enzyme activities, and differentially expressed genes and miRNAs involved in the carbon and nitrogen metabolic pathway in rice shoots at the following times: 2:00, 6:00, 10:00, 14:00, 18:00, and 22:00. Our results indicated that more CO2 was fixed into carbohydrates by a high net photosynthetic rate, respiratory rate, and stomatal conductance in the daytime. Although high levels of the nitrate reductase activity, free ammonium and carbohydrates were exhibited in the daytime, the protein synthesis was not significantly facilitated by the light and temperature. In mRNA sequencing, the carbon and nitrogen metabolism-related differentially expressed genes were obtained, which could be divided into eight groups: photosynthesis, TCA cycle, sugar transport, sugar metabolism, nitrogen transport, nitrogen reduction, amino acid metabolism, and nitrogen regulation. Additionally, a total of 78,306 alternative splicing events have been identified, which primarily belong to alternative 5′ donor sites, alternative 3′ acceptor sites, intron retention, and exon skipping. In sRNA sequencing, four carbon and nitrogen metabolism-related miRNAs (osa-miR1440b, osa-miR2876-5p, osa-miR1877 and osa-miR5799) were determined to be regulated by natural light/dark cycle. The expression level analysis showed that the four carbon and nitrogen metabolism-related miRNAs negatively regulated their target genes. These results may provide a good strategy to study how natural light/dark cycle regulates carbon and nitrogen metabolism to ensure plant growth and development.

Genetic analysis of seed mineral accumulation affected by phosphorus deprivation in Brassica napus

The mineral content of plant seeds depends on both environmental and genetic factors. The aim of this study was to detect quantitative trait loci (QTLs), and their candidate genes, for the accumulation of phosphorus (P), calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), iron (Fe), and manganese (Mn) in seeds of Brassica napus under normal and low P conditions using an F10 recombinant inbred line (RIL) population. Two-year field trials were conducted to investigate seed mineral accumulation. The results showed a significant decrease in most of the minerals in the BE RIL population, as well as in two parental lines, when grown in a low P environment compared to a normal P environment. In total, 60 putative QTLs were identified, 33 of which overlapped with each other in nine genomic regions in seven linkage groups. Twenty-one of the 60 significant QTLs co-located with eight seed weight QTLs, and only five overlapped with three seed yield QTLs. Moreover, only six QTLs for the same minerals were identified both in normal and low P levels. By comparative mapping of Arabidopsis and B. napus, 148 orthologs of 97 genes involved in the homeostasis of the seven minerals in Arabidopsis were associated with 47 QTLs corresponding to 24 chromosomal regions. These results offer insight into the genetic basis of mineral accumulation across different P conditions in seeds of B. napus and allow the potential utilization of QTLs in biofortification.

Breeding histories and selection criteria for oilseed rape in Europe and China identified by genome wide pedigree dissection

Selection breeding has played a key role in the improvement of seed yield and quality in oilseed rape (Brassica napus L.). We genotyped Tapidor (European), Ningyou7 (Chinese) and their progenitors with the Brassica 60 K Illumina Infinium SNP array and mapped a total of 29,347 SNP markers onto the reference genome of Darmor-bzh. Identity by descent (IBD) refers to a haplotype segment of a chromosome inherited from a shared common ancestor. IBDs identified on the C subgenome were larger than those on the A subgenome within both the Tapidor and Ningyou7 pedigrees. IBD number and length were greater in the Ningyou7 pedigree than in the Tapidor pedigree. Seventy nine QTLs for flowering time, seed quality and root morphology traits were identified in the IBDs of Tapidor and Ningyou7. Many more candidate genes had been selected within the Ningyou7 pedigree than within the Tapidor pedigree. These results highlight differences in the transfer of favorable gene clusters controlling key traits during selection breeding in Europe and China.

Genetic variants associated with the root system architecture of oilseed rape (Brassica napus L.) under contrasting phosphate supply

Abstract Breeding crops with ideal root system architecture for efficient absorption of phosphorus is an important strategy to reduce the use of phosphate fertilizers. To investigate genetic variants leading to changes in root system architecture, 405 oilseed rape cultivars were genotyped with a 60K Brassica Infinium SNP array in low and high P environments. A total of 285 single-nucleotide polymorphisms were associated with root system architecture traits at varying phosphorus levels. Nine single-nucleotide polymorphisms corroborate a previous linkage analysis of root system architecture quantitative trait loci in the BnaTNDH population. One peak single-nucleotide polymorphism region on A3 was associated with all root system architecture traits and co-localized with a quantitative trait locus for primary root length at low phosphorus. Two more single-nucleotide polymorphism peaks on A5 for root dry weight at low phosphorus were detected in both growth systems and co-localized with a quantitative trait locus for the same trait. The candidate genes identified on A3 form a haplotype ‘BnA3Hap’, that will be important for understanding the phosphorus/root system interaction and for the incorporation into Brassica napus breeding programs.