SlTCP24 and SlTCP29 synergistically regulate compound leaf development through interacting with SlAS2 and activating transcription of SlCKX2 in tomato.

The complexity of compound leaves results primarily from the leaflet initiation and arrangement during leaf development. However, the molecular mechanism underlying compound leaf development remains a central research question. SlTCP24 and SlTCP29, two plant-specific transcription factors with the conserved TCP motif, are shown here to synergistically regulate compound leaf development in tomato. When both of them were knocked out simultaneously, the number of leaflets significantly increased, and the shape of the leaves became more complex. SlTCP24 and SlTCP29 could form both homodimers and heterodimers, and such dimerization was impeded by the leaf polarity regulator SlAS2, which interacted with SlTCP24 and SlTCP29. SlTCP24 and SlTCP29 could bind to the TCP-binding cis-element of the SlCKX2 promoter and activate its transcription. Transgenic plants with SlTCP24 and SlTCP29 double-gene knockout had a lowered transcript level of SlCKX2 and an elevated level of cytokinin. This work led to the identification of two key regulators of tomato compound leaf development and their targeted genes involved in cytokinin metabolic pathway. A model of regulation of compound leaf development was proposed based on observations of this study.

Auxin Biosynthesis Genes in Allotetraploid Oilseed Rape Are Essential for Plant Development and Response to Drought Stress.

Crucial studies have verified that IAA is mainly generated via the two-step pathway in Arabidopsis, in which tryptophan aminotransferase (TAA) and YUCCA (YUC) are the two crucial enzymes. However, the role of the TAA (or TAR) and YUC genes in allotetraploid oilseed rape underlying auxin biosynthesis and development regulation remains elusive. In the present study, all putative TAR and YUC genes were identified in B. napus genome. Most TAR and YUC genes were tissue that were specifically expressed. Most YUC and TAR proteins contained trans-membrane regions and were confirmed to be endoplasmic reticulum localizations. Enzymatic activity revealed that YUC and TAR protein members were involved in the conversion of IPA to IAA and Trp to IPA, respectively. Transgenic plants overexpressing BnaYUC6a in both Arabidopsis and B. napus displayed high auxin production and reduced plant branch angle, together with increased drought resistance. Moreover, mutation in auxin biosynthesis BnaTARs genes by CRISPR/Cas9 caused development defects. All these results suggest the convergent role of BnaYUC and BnaTAR genes in auxin biosynthesis. Different homoeologs of BnaYUC and BnaTAR may be divergent according to sequence and expression variation. Auxin biosynthesis genes in allotetraploid oilseed rape play a pivotal role in coordinating plant development processes and stress resistance.

Conservation and Divergence of the CONSTANS-Like (COL) Genes Related to Flowering and Circadian Rhythm in Brassica napus.

The CONSTANS-LIKE (COL) genes are important signaling component in the photoperiod pathway and flowering regulation pathway. However, people still know little about their role in Brassica napus. To achieve a better understanding of the members of the BnaCOL gene family, reveal their evolutionary relationship and related functions involved in photoperiod regulation, we systematically analyzed the BnaCOL family members in B. napus genome. A total of 33 BnaCOL genes distributed unevenly on 16 chromosomes were identified in B. napus and could be classified into three subfamilies. The same subfamilies have relatively conservative gene structures, three-dimensional protein structures and promoter motifs such as light-responsive cis-elements. The collinearity analysis detected 37 pairs of repetitive genes in B. napus genome. A 67.7% of the BnaCOL genes were lost after B. napus genome polyploidization. In addition, the BnaCOL genes showed different tissue-specific expression patterns. A 81.8% of the BnaCOL genes were mainly expressed in leaves, indicating that they may play a conservative role in leaves. Subsequently, we tested the circadian expression profiles of nine homologous genes that regulate flowering in Arabidopsis. Most BnaCOL genes exhibit several types of circadian rhythms, indicating that these BnaCOL genes are involved in the photoperiod pathway. As such, our research has laid the foundation for understanding the exact role of the BnaCOL family in the growth and development of rapeseed, especially in flowering.

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.

A copia-like retrotransposon insertion in the upstream region of the SHATTERPROOF1 gene, BnSHP1.A9, is associated with quantitative variation in pod shattering resistance in oilseed rape.

Seed loss resulting from pod shattering is a major constraint in production of oilseed rape (Brassica napus L.). However, the molecular mechanisms underlying pod shatter resistance are not well understood. Here, we show that the pod shatter resistance at quantitative trait locus qSRI.A9.1 is controlled by one of the B. napus SHATTERPROOF1 homologs, BnSHP1.A9, in a doubled haploid population generated from parents designated R1 and R2 as well as in a diverse panel of oilseed rape. The R1 maternal parental line of the doubled haploid population carried the allele for shattering at qSRI.A9.1, while the R2 parental line carried the allele for shattering resistance. Quantitative RT-PCR showed that BnSHP1.A9 was expressed specifically in flower buds, flowers, and developing siliques in R1, while it was not expressed in any tissue of R2. Transgenic plants constitutively expressing either of the BnSHP1.A9 alleles from the R1 and R2 parental lines showed that both alleles are responsible for pod shattering, via a mechanism that promotes lignification of the enb layer. These findings indicated that the allelic differences in the BnSHP1.A9 gene per se are not the causal factor for quantitative variation in shattering resistance at qSRI.A9.1. Instead, a highly methylated copia-like long terminal repeat retrotransposon insertion (4803 bp) in the promotor region of the R2 allele of BnSHP1.A9 repressed the expression of BnSHP1.A9, and thus contributed to pod shatter resistance. Finally, we showed a copia-like retrotransposon-based marker, BnSHP1.A9R2, can be used for marker-assisted breeding targeting the pod shatter resistance trait in oilseed rape.

Integrative RNA- and miRNA-Profile Analysis Reveals a Likely Role of BR and Auxin Signaling in Branch Angle Regulation of B. napus.

Oilseed rape (Brassica napus L.) is the second largest oilseed crop worldwide and one of the most important oil crops in China. As a component of plant architecture, branch angle plays an important role in yield performance, especially under high-density planting conditions. However, the mechanisms underlying the regulation of branch angle are still largely not understood. Two oilseed rape lines with significantly different branch angles were used to conduct RNA- and miRNA-profiling at two developmental stages, identifying differential expression of a large number of genes involved in auxin- and brassinosteroid (BR)-related pathways. Many auxin response genes, including AUX1, IAA, GH3, and ARF, were enriched in the compact line. However, a number of genes involved in BR signaling transduction and biosynthesis were down-regulated. Differentially expressed miRNAs included those involved in auxin signaling transduction. Expression patterns of most target genes were fine-tuned by related miRNAs, such as miR156, miR172, and miR319. Some miRNAs were found to be differentially expressed at both developmental stages, including three miR827 members. Our results provide insight that auxin- and BR-signaling may play a pivotal role in branch angle regulation.

Identification of BnaYUCCA6 as a candidate gene for branch angle in Brassica napus by QTL-seq.

Oilseed rape (Brassica napus L.) is one of the most important oil crops in China as well as worldwide. Branch angle as a plant architecture component trait plays an important role for high density planting and yield performance. In this study, bulked segregant analysis (BSA) combined with next generation sequencing technology was used to fine map QTL for branch angle. A major QTL, designated as branch angle 1 (ba1) was identified on A06 and further validated by Indel marker-based classical QTL mapping in an F2 population. Eighty-two genes were identified in the ba1 region. Among these genes, BnaA0639380D is a homolog of AtYUCCA6. Sequence comparison of BnaA0639380D from small- and big-branch angle oilseed rape lines identified six SNPs and four amino acid variation in the promoter and coding region, respectively. The expression level of BnaA0639380D is significantly higher in the small branch angle line Purler than in the big branch angle line Huyou19, suggesting that the genomic mutations may result in reduced activity of BnaA0639380D in Huyou19. Phytohormone determination showed that the IAA content in Purler was also obviously increased. Taken together, our results suggested BnaA0639380D is a possible candidate gene for branch angle in oilseed rape.

Multigenic Control of Pod Shattering Resistance in Chinese Rapeseed Germplasm Revealed by Genome-Wide Association and Linkage Analyses.

The majority of rapeseed cultivars shatter seeds upon maturity especially under hot-dry and windy conditions, reducing yield and gross margin return to growers. Here, we identified quantitative trait loci (QTL) for resistance to pod shatter in an unstructured diverse panel of 143 rapeseed accessions, and two structured populations derived from bi-parental doubled haploid (DH) and inter-mated (IF2) crosses derived from R1 (resistant to pod shattering) and R2 (prone to pod shattering) accessions. Genome-wide association analysis identified six significant QTL for resistance to pod shatter located on chromosomes A01, A06, A07, A09, C02, and C05. Two of the QTL, qSRI.A09 delimited with the SNP marker Bn-A09-p30171993 (A09) and qSRI.A06 delimited with the SNP marker Bn-A06-p115948 (A06) could be repeatedly detected across environments in a diversity panel, DH and IF2 populations, suggesting that at least two loci on chromosomes A06 and A09 were the main contributors to pod shatter resistance in Chinese germplasm. Significant SNP markers identified in this study especially those that appeared repeatedly across environments provide a cost-effective and an efficient method for introgression and pyramiding of favorable alleles for pod shatter resistance via marker-assisted selection in rapeseed improvement programs.