A matter of time: regulatory events behind the synchronization of C-4 and crassulacean acid metabolism in Portulaca oleracea

Portulaca species can switch between C4 and crassulacean acid metabolism (CAM) depending on environmental conditions. However, the regulatory mechanisms behind this rare photosynthetic adaptation remain elusive. Using Portulaca oleracea as a model system, here we investigated the involvement of the circadian clock, plant hormones, and transcription factors in coordinating C4 and CAM gene expression. Free-running experiments in constant conditions suggested that C4 and CAM gene expression are intrinsically connected to the circadian clock. Detailed time-course, drought, and rewatering experiments revealed distinct time frames for CAM induction and reversion (days versus hours, respectively), which were accompanied by changes in abscisic acid (ABA) and cytokinin metabolism and signaling. Exogenous ABA and cytokinins were shown to promote and repress CAM expression in P. oleracea, respectively. Moreover, the drought-induced decline in C4 transcript levels was completely recovered upon cytokinin treatment. The ABA-regulated transcription factor genes HB7, NFYA7, NFYC9, TT8, and ARR12 were identified as likely candidate regulators of CAM induction following this approach, whereas NFYC4 and ARR9 were connected to C4 expression patterns. Therefore, we provide insights into the signaling events controlling C4-CAM transitions in response to water availability and over the day/night cycle, highlighting candidate genes for future functional studies in the context of facultative C4-CAM photosynthesis

Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

<div><p>Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between <i>Saccharum officinarum</i> and <i>Saccharum spontaneum</i>. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100–130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for <i>Saccharum officinarum</i>, <i>Saccharum spontaneum</i>, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation.</p></div

Summary of ORFeome construction and main results.

<p>The dashed line denotes future directions for sugarcane ORFeome studies. This ORFeome can be used for gene discovery related to a range of traits since over 5,000 different categories (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107351#pone.0107351.s006" target="_blank">Table S4</a>) have a full-length representative. Differentially expressed alleles in each sample and in the hybrid and their origin from each ancestral genotype can also be analyzed. Several types of polymorphisms and genetic variability can be further investigated. Both genome assembly and annotation can make use of this sugarcane ORFeome dataset to validate and improve results. TF, transcription factor; NAT, natural antisense transcript.</p

Top four categories of genotype-specific contigs based on Phytozome annotations.

<p>Only contigs from leaf samples in each genotype were considered.</p

Number of contigs and percentage of coverage by antisense reads.

<p>Non-overlapping contig coverage by antisense reads was calculated for all contigs showing antisense expression (28,844 contigs).</p

Data from Ion PGM and 454 sequencing (after trimming) and Trinity assembly output.

<p>Data from Ion PGM and 454 sequencing (after trimming) and Trinity assembly output.</p

Description of cloned full-length cDNA libraries.

a<p>Excluding low-quality sequences and repeated clones.</p>b<p>Excluding sugarcane-specific transcripts.</p><p>Description of cloned full-length cDNA libraries.</p

Number of reads identified as natural antisense transcripts (NATs), based on their orientation on alignment to grass genes.

<p>Tissues are indicated as follows: In1, immature internodes; In5, intermediate internodes; L, leaves.</p

Length distribution of sugarcane ORFs, full-length transcripts (FL), and UTRs.

<p>Graphs denote the comparison of sugarcane length distribution (gray bars) of ORFs (A) and full-length transcripts (B) to other grasses (colored lines). Length distribution of 5′ and 3′ UTRs (C, black and white bars, respectively) of sugarcane full-length transcripts is shown as well.</p