Transcriptome analysis of oil palm inflorescences revealed candidate genes for an auxin signaling pathway involved in parthenocarpy

Oil palm parthenocarpic fruits, which are produced without fertilization, can be targeted to increase oil content because the majority of the fruit is occupied by mesocarp, the part in which palm oil is stored. Consequently, gaining an understanding of the parthenocarpic mechanism would be instrumental for producing parthenocarpic oil palm. This study aims to determine effects of auxin treatment and analyze differentially expressed genes in oil palm pistils at the pollination/anthesis stage, using an RNA sequencing (RNA seq) approach. The auxin treatment caused 100% parthenocarpy when auxin was sprayed before stigmas opened. The parthenocarpy decreased to 55%, 8% and 5% when the auxin was sprayed 1, 2 and 3 days after the opening of stigmas, respectively. Oil palm plants used for RNA seq were plants untreated with auxin as controls and auxin-treated plants on the day before pollination and 1 day after pollination. The number of raw reads ranged from 8,425,859 to 11,811,166 reads, with an average size ranging from 99 to 137 base pairs (bp). When compared with the oil palm transcriptome, the mapped reads ranged from 8,179,948 to 11,320,799 reads, representing 95.85–98.01% of the oil palm matching. Based on five comparisons between RNA seq of treatments and controls, and confirmation using reverse transcription polymerase chain reaction and quantitative real-time RT-PCR expression, five candidate genes, including probable indole-3-acetic acid (IAA)-amido synthetase GH3.8 (EgGH3.8), IAA-amido synthetase GH3.1 (EgGH3.1), IAA induced ARG7 like (EgARG7), tryptophan amino transferase-related protein 3-like (EgTAA3) and flavin-containing monooxygenase 1 (EgFMO1), were differentially expressed between auxin-treated and untreated samples. This evidence suggests a pathway of parthenocarpic fruit development at the beginning of fruit development. However, more research is needed to identify which genes are definitely involved in parthenocarpy

Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing

Background Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs. Methods We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors. Results A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants. Discussion The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar

The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

Identification and Regulation of Plasma Membrane Sulfate Transporters in Chlamydomonas1[W][OA]

Chlamydomonas (Chlamydomonas reinhardtii) exhibits several responses following exposure to sulfur (S)-deprivation conditions, including an increased efficiency of import and assimilation of the sulfate anion (SO42−). Aspects of SO42− transport during S-replete and S-depleted conditions were previously studied, although the transporters had not been functionally identified. We employed a reverse genetics approach to identify putative SO42− transporters, examine their regulation, establish their biogenesis and subcellular locations, and explore their functionality. Upon S starvation of wild-type Chlamydomonas cells, the accumulation of transcripts encoding the putative SO42− transporters SLT1 (for SAC1-like transporter 1), SLT2, and SULTR2 markedly increased, suggesting that these proteins function in high-affinity SO42− transport. The Chlamydomonas sac1 and snrk2.1 mutants (defective for acclimation to S deprivation) exhibited much less of an increase in the levels of SLT1, SLT2, and SULTR2 transcripts and their encoded proteins in response to S deprivation compared with wild-type cells. All three transporters were localized to the plasma membrane, and their rates of turnover were significantly impacted by S availability; the turnover of SLT1 and SLT2 was proteasome dependent, while that of SULTR2 was proteasome independent. Finally, mutants identified for each of the S-deprivation-responsive transporters were used to establish their critical role in the transport of SO42− into S-deprived cells

Identification of Candidate Gene-Based Markers for Girth Growth in Rubber Trees

Girth growth is an important factor in both latex and timber production of the rubber tree. In this study, we performed candidate gene association mapping for girth growth in rubber trees using intron length polymorphism markers (ILP) in identifying the candidate genes responsible for girth growth. The COBL064_1 marker developed from the candidate gene (COBL4) regulating cellulose deposition and oriented cell expansion in the plant cell wall showed the strongest association with girth growth across two seasons in the Amazonian population and was validated in the breeding lines. We then applied single molecule real-time (SMRT) circular consensus sequencing (CCS) to analyze a wider gene region of the COBL4 to pinpoint the single nucleotide polymorphism (SNP) that best explains the association with the traits. A SNP in the 3’ UTR showing linkage disequilibrium with the COBL064_1 most associated with girth growth. This study showed that the cost-effective method of ILP gene-based markers can assist in identification of SNPs in the candidate gene associated with girth growth. The SNP markers identified in this study added useful markers for the improvement of girth growth in rubber tree breeding programs

The Central Role of a SNRK2 Kinase in Sulfur Deprivation Responses1[W][OA]

In the absence of sulfur (S), Chlamydomonas reinhardtii increases the abundance of several transcripts encoding proteins associated with S acquisition and assimilation, conserves S amino acids, and acclimates to suboptimal growth conditions. A positive regulator, SAC1 (for sulfur acclimation protein 1), and a negative regulator, SAC3, were shown to participate in the control of these processes. In this study, we investigated two allelic mutants (ars11 and ars44) affected in a gene encoding a SNRK2 (for SNF1-related protein kinase 2) kinase designated SNRK2.1. Like the sac1 mutant, both snrk2.1 mutants were deficient in the expression of S-responsive genes. Furthermore, the mutant cells bleached more rapidly than wild-type cells during S deprivation, although the phenotypes of ars11 and ars44 were not identical: ars11 exhibited a more severe phenotype than either ars44 or sac1. The phenotypic differences between the ars11 and ars44 mutants reflected distinct alterations of SNRK2.1 mRNA splicing caused by insertion of the marker gene. The ars11 phenotype could be rescued by complementation with SNRK2.1 cDNA. In contrast to the nonepistatic relationship between SAC3 and SAC1, characterization of the sac3 ars11 double mutant showed that SNRK2.1 is epistatic to SAC3. These data reveal the crucial regulatory role of SNRK2.1 in the signaling cascade critical for eliciting S deprivation responses in Chlamydomonas. The phylogenetic relationships and structures of the eight members of the SNRK2 family in Chlamydomonas are discussed