Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins.

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system.

CRISPR/Cas-base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A-PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base-editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base-edited plants of ALS conferred high herbicide resistance, while base-edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T0 to T1 generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off-target sites, large-scale SNP variations were determined through whole-genome sequencing between some base-edited and wild-type plants. These results revealed that A3A-PBE base-editing system could effectively convert C to T substitution with high-editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.

Identification of Bna.IAA7.C05 as allelic gene for dwarf mutant generated from tissue culture in oilseed rape.

BACKGROUND: Plant height is one of the most important agronomic traits in many crops due to its influence on lodging resistance and yield performance. Although progress has been made in the use of dwarfing genes in crop improvement, identification of new dwarf germplasm is still of significant interest for breeding varieties with increased yield. RESULTS: Here we describe a dominant, dwarf mutant G7 of Brassica napus with down-curved leaves derived from tissue culture. To explore the genetic variation responsible for the dwarf phenotype, the mutant was crossed to a conventional line to develop a segregating F2 population. Bulks were formed from plants with either dwarf or conventional plant height and subjected to high throughput sequencing analysis via mutation mapping (MutMap). The dwarf mutation was mapped to a 0.6 Mb interval of B. napus chromosome C05. Candidate gene analysis revealed that one SNP causing an amino acid change in the domain II of Bna.IAA7.C05 may contribute to the dwarf phenotype. This is consistent with the phenotype of a gain-of-function indole-3-acetic acid (iaa) mutant in Bna.IAA7.C05 reported recently. GO and KEGG analysis of RNA-seq data revealed the down-regulation of auxin related genes, including many other IAA and small up regulated response (SAUR) genes, in the dwarf mutant. CONCLUSION: Our studies characterize a new allele of Bna.IAA7.C05 responsible for the dwarf mutant generated from tissue culture. This may provide a valuable genetic resource for breeding for lodging resistance and compact plant stature in B. napus.

Transcriptome and Hormone Comparison of Three Cytoplasmic Male Sterile Systems in Brassica napus.

The interaction between plant mitochondria and the nucleus markedly influences stress responses and morphological features, including growth and development. An important example of this interaction is cytoplasmic male sterility (CMS), which results in plants producing non-functional pollen. In current research work, we compared the phenotypic differences in floral buds of different Brassica napus CMS (Polima, Ogura, Nsa) lines with their corresponding maintainer lines. By comparing anther developmental stages between CMS and maintainer lines, we identified that in the Nsa CMS line abnormality occurred at the tetrad stage of pollen development. Phytohormone assays demonstrated that IAA content decreased in sterile lines as compared to maintainer lines, while the total hormone content was increased two-fold in the S2 stage compared with the S1 stage. ABA content was higher in the S1 stage and exhibited a two-fold decreasing trend in S2 stage. Sterile lines however, had increased ABA content at both stages compared with the corresponding maintainer lines. Through transcriptome sequencing, we compared differentially expressed unigenes in sterile and maintainer lines at both (S1 and S2) developmental stages. We also explored the co-expressed genes of the three sterile lines in the two stages and classified these genes by gene function. By analyzing transcriptome data and validating by RT-PCR, it was shown that some transcription factors (TFs) and hormone-related genes were weakly or not expressed in the sterile lines. This research work provides preliminary identification of the pollen abortion stage in Nsa CMS line. Our focus on genes specifically expressed in sterile lines may be useful to understand the regulation of CMS.

A fluorescence-based high-throughput screening method for cytokinin translocation mutants.

BACKGROUND: Cytokinins are one kind of phytohormones essential for plant growth, development and stress responses. In the past half century, significant progresses have been made in the studies of cytokinin signal transduction and metobolic pathways, but the mechanism of cytokinin translocation is poorly understood. Arabidopsis (Arabidopsis thaliana) response regulator 5 (ARR5) is a type-A response factor in cytokinin signaling which is induced by cytokinins and has been used as a reporter gene for the endogenous cytokinins in Arabidopsis. Here, we report a fluorescence-based high-throughput method to screen cytokinin translocation mutants using an ethyl methyl sulfone (EMS) mutagenesis library generated with ARR5::eGFP transgenic plants. RESULTS: The seedlings with enhanced green fluorescent protein (GFP) signal in roots were screened in a luminescence imaging system (LIS) in large scale to obtain mutants with over-accumulated cytokinins in roots. The selected mutants were confirmed under a fluorescence microscopy and then performed phenotypic analysis. In this way, we obtained twelve mutants with elevated GFP signal in the roots and further found three of them displayed reduced GFP signal in the aerial tissues. Two of the mutants were characterized and proved to be the atabcg14 allelic mutants which are defective in the long-distance translocation of root-synthesized cytokinins. CONCLUSIONS: We provide a strategy for screening mutants defective in cytokinin translocation, distribution or signaling. The strategy can be adapted to establish a system for screening mutants defective in other hormone transporters or signaling components using a fluorescence reporter.

QTL and Candidate Gene Identification for Silique Length Based on High-Dense Genetic Map in Brassica napus L.

Silique length (SL) is an important yield trait and positively correlates with seeds per silique and seed weight. In the present study, two double haploid (DH) populations, established from crosses Zhongshuang11 × R11 (ZR) and R1 × R2 (RR), containing 280 and 95 DH lines, respectively, were used to map quantitative trait loci (QTL) for SL. A high-dense genetic map from ZR population was constructed comprising 14,658 bins on 19 linkage groups, with map length of 2,198.85 cM and an average marker distance of 0.15 cM. Genetic linkage map from RR population was constructed by using 2,046 mapped markers anchored to 19 chromosomes with 2,217-cM map length and an average marker distance of 1.08 cM. Major QTL qSL_ZR_A09 and qSL_RR_A09b on A09 were identified from ZR and RR populations, respectively. Both QTL could be stably detected in four environments. QTL qSL_RR_A09b and qSL_ZR_A09 were located on 68.5-70.8 cM and 91.33-91.94 cM interval with R2 values of 14.99-39.07% and 15.00-20.36% in RR and ZR populations, respectively. Based on the physical positions of single nucleotide polymorphism (SNP) markers flanking qSL_ZR_A09 and gene annotation in Arabidopsis, 26 genes were identified with SNP/Indel variation between parents and two genes (BnaA09g41180D and BnaA09g41380D) were selected as the candidate genes. Expression analysis further revealed BnaA09g41180D, encoding homologs of Arabidopsis fasciclin-like arabinogalactan proteins (FLA3), as the most promising candidate gene for qSL_ZR_A09. The QTL identification and candidate gene analysis will provide new insight into the genomic regions controlling SL in Brassica napus as well as candidate genes underlying the QTL.