Analysis of ambient temperature-responsive transcriptome in shoot apical meristem of heat-tolerant and heat-sensitive broccoli inbred lines during floral head formation.

BACKGROUND: Head formation of broccoli (Brassica oleracea var. italica) is greatly reduced under high temperature (22 °C and 27 °C). Broccoli inbred lines that are capable of producing heads at high temperatures in summer are varieties that are unique to Taiwan. However, knowledge of the early-activated pathways of broccoli head formation under high temperature is limited. RESULTS: We compared heat-tolerant (HT) and heat-sensitive (HS) transcriptome of broccoli under different temperatures. Weighted gene correlation network analysis (WGCNA) revealed that genes involved in calcium signaling pathways, mitogen-activated protein kinase (MAPK) cascades, leucine-rich repeat receptor-like kinases (LRR-RLKs), and genes coding for heat-shock proteins and reactive oxygen species homeostasis shared a similar expression pattern to BoFLC1, which was highly expressed at high temperature (27 °C). Of note, these genes were less expressed in HT than HS broccoli at 22 °C. Co-expression analysis identified a model for LRR-RLKs in survival-reproduction tradeoffs by modulating MAPK- versus phytohormones-signaling during head formation. The difference in head-forming ability in response to heat stress between HT and HS broccoli may result from their differential transcriptome profiles of LRR-RLK genes. High temperature induced JA- as well as suppressed auxin- and cytokinin-related pathways may facilitate a balancing act to ensure fitness at 27 °C. BoFLC1 was less expressed in HT than HS at 22 °C, whereas other FLC homologues were not. Promoter analysis of BoFLC1 showed fewer AT dinucleotide repeats in HT broccoli. These results provide insight into the early activation of stress- or development-related pathways during head formation in broccoli. The identification of the BoFLC1 DNA biomarker may facilitate breeding of HT broccoli. CONCLUSIONS: In this study, HT and HS broccoli genotypes were used to determine the effect of temperature on head formation by transcriptome profiling. On the basis of the expression pattern of high temperature-associated signaling genes, the HS transcriptome may be involved in stress defense instead of transition to the reproductive phase in response to heat stress. Transcriptome profiling of HT and HS broccoli helps in understanding the molecular mechanisms underlying head-forming capacity and in promoting functional marker-assisted breeding.

Comparative miRNAs analysis of Two contrasting broccoli inbred lines with divergent head-forming capacity under temperature stress.

BACKGROUND: MicroRNAs (miRNAs) play a vital role in growth, development, and stress response at the post-transcriptional level. Broccoli (Brassica oleracea L. var italic) is an important vegetable crop, and the yield and quality of broccoli are decreased by heat stress. The broccoli inbred lines that are capable of producing head at high temperature in summer are unique varieties in Taiwan. However, knowledge of miRNAomes during the broccoli head formation under heat stress is limited. METHODS: In this study, molecular characterization of two nearly isogenic lines with contrasting head-forming capacity was investigated. Head-forming capacity was better for heat-tolerant (HT) than heat-sensitive (HS) broccoli under heat stress. RESULTS: By deep sequencing and computational analysis, 20 known miRNAs showed significant differential expression between HT and HS genotypes. According to the criteria for annotation of new miRNAs, 24 novel miRNA sequences with differential expression between the two genotypes were identified. To gain insight into functional significance, 213 unique potential targets of these 44 differentially expressed miRNAs were predicted. These targets were implicated in shoot apical development, phase change, response to temperature stimulus, hormone and energy metabolism. The head-forming capacity of the unique HT line was related to autonomous regulation of Bo-FT genes and less expression level of heat shock protein genes as compared to HS. For the genotypic comparison, a set of miRNAs and their targets had consistent expression patterns in various HT genotypes. CONCLUSIONS: This large-scale characterization of broccoli miRNAs and their potential targets is to unravel the regulatory roles of miRNAs underlying heat-tolerant head-forming capacity.

The influence of adjacent nucleotides on the pattern of nucleotide substitution in mitochondrial introns of angiosperms.

It has been known that in noncoding regions of the chloroplast genome, the pattern of nucleotide substitution is influenced by the two nucleotides flanking the substitution site. In a GC-rich environment, a bias toward transition was observed, whereas in an AT-rich environment, a bias toward transversion was observed. In this study, the influence of the two adjacent neighbors on the substitution pattern was observed in the first intron of the mitochondrial nad4 gene, although the AT content of this intron is only 48%. The proportion of transversions increases from 0.32 to 0.75 as the A + T content (number of A's + T's) of the two nearest neighbors increases from 0 to 2. This trend was also observed in another mitochondrial group I intron with an AT content of 64%. In addition, a similar, though weaker, effect was observed in vertebrate pseudogenes. So this effect is present in all three types of genomes. Furthermore, in contrast to the situation in the noncoding regions of chloroplast DNA, where most nucleotide substitutions occurred in the categories with an A + T content of either 1 or 2, nucleotide substitutions in the mitochondrial first nad4 intron occurred more evenly in three categories of different A + T contents. This might be due largely to the difference in the AT content (0.48 vs. 0.72) between the mitochondrial first nad4 intron and the chloroplast DNA regions studied.

A study of the phylogeny of Brassica rapa, B. nigra, Raphanus sativus, and their related genera using noncoding regions of chloroplast DNA.

There are two evolutionary lineages in the genus Brassica: the rapa/oleracea lineage and the nigra lineage. Using nuclear DNA sequences such as the intergenic spacer between 5S rRNA genes and the internal transcribed spacer between 18S and 25S rRNA genes, we and others had previously demonstrated that Raphanus sativus is closely related to the nigra lineage. In the present study, we sequenced the chloroplast noncoding region between trnT and trnF and that between trnD and trnT in seven species and showed that R. sativus is more closely related to the rapa/oleracea lineage than to the nigra lineage. The conflicting results from nuclear DNA and chloroplast DNA support the hypothesis that Raphanus was derived from a hybridization between the rapa/oleracea and the nigra lineages. We estimated the date of this hybridization event to be 60% of the divergence time between the two Brassica lineages. In addition, the pattern and rate of nucleotide substitution were studied. There were more transversions than transitions in these noncoding regions, which have a high AT content. Furthermore, the proportion of transversions among the substitutions at a site increases with increasing A + T content of its two adjacent nucleotides. An influence of immediate 5(') pyrimidine on substitution pattern is also observed when both adjacent bases in the two DNA strands are A or T. The rate of nucleotide substitution in the trnL group I intron is only about one third of the rate in the nearby intergenic spacers in the trnT-trnF fragment. The rate of nucleotide substitution in the rapa/oleracea lineage is at least 1.5 times that in the nigra lineage.