Liquid biofuels, such as bioethanol, converted from biomass are considered as a promising alternative for traditional fossil fuels. Biofuels development can reduce greenhouse gas emission and meet the world's rapidly growing demand for energy. Currently, bioethanol is mainly produced from feedstocks with relatively high starch or sugar percentage, such as corn, sugarcane, sweet potato and cassava [1–3]. However, these bioethanol production modes have some inherent problems, including the adverse impacts on food security, environment and insufficient agricultural land [4, 5]. Although lignocellulosic sources are also considered as a promising feedstock for bioethanol production, there are several obstacles, such as the lack of an efficient, economical and environment friendly pretreatment process, that still needed to be overcome . Therefore, developing sustainable feedstocks and processing protocols for biofuel production is becoming more and more urgent.
One alternative feedstock is duckweed (Lemnacecae family), a small flowering plant that has a global adaptability across a broad range of climates [7, 8] and can be easily found in quiescent or slowly flowing and polluted water bodies worldwide . Duckweed has a longer yearly production period than most of other plants, and even grows year-round in some areas with a warm climate , which make it a potential sustainable feedstock for industrial application. Previous studies indicated that this plant produces biomass faster than any other flowering plant . With near-exponential growth rates and the shortest doubling times [12–14], duckweed can achieve a biomass of 0.5 to 1.5 metric tons/hectare/day fresh weight or 13 to 38 metric tons/hectare/year dry weight . This accompanies with the tremendous amount of CO2 sequestered from the atmosphere and the natural ability of duckweed to thrive on eutrophic wastewater, and recover polluting nutrients [14, 16–23], suggesting that growing duckweed for biomass can have large beneficial environmental impacts. In warm seasons, duckweed can remove up to 85% of total Kjehldahl nitrogen (TKN) and 78% of total phosphorous (TP) . Importantly, duckweed biomass exhibits good characteristics for bioethanol production due to its relatively high starch and low lignin percentage [24–27]. High starch accumulation is the most important trait of this crop. Depending on the duckweed species and growing conditions, starch percentage of duckweed ranges from 3% to 75% (Dry weight, DW) . Under nutrient-rich growth conditions, duckweed has a relatively low starch percentage. But by manipulating growing conditions, including the adjustment of pH, phosphate concentration, and nutrient status, starch percentage of duckweed can be significantly increased [26, 27, 29]. It is encouraging that published studies have demonstrated that carbohydrate derived from duckweed could be converted to ethanol and butanol efficiently [26, 27, 30–32]. As a novel feedstock for bioenergy production, duckweed has recently gained interest from researchers and governments. In 2009, the United States Department of Energy supported a project to sequence the genome of Spirodela polyrhiza (CSP2009, CSP_LOI_793583). Supported by the Minister of Science and Technology, China launched a project to produce liquid biofuel from duckweed biomass, and the first international conference on duckweed application and research was held in Chengdu, China in October, 2011 .
Duckweed was once an important model system for plant biology before the days of Arabidopsis[33–36] and facilitated important advances in plant biology, such as contributions to our understanding of photoperiod control of flowering [34, 37, 38], sulfur metabolic pathways and auxin biosynthesis . With the advent of modern genetics and genomics, the status of duckweed as model plant was replaced by other plants, such as Arabidopsis and rice, mainly because the exceedingly tiny size and infrequent flowers made genetics studies and breeding in duckweed difficult. With its new value rediscovered, molecular research on duckweed has been carried out again. Several whole chloroplast genome data of duckweed were released [39, 40], nuclear genome size were measured by flow cytometry (FCM) , and DNA barcoding technology was developed to distinguish different species in Lemnacecae family [42–44]. However, as a potential energy crop, functional genomics and transcriptomics data for duckweed are urgently needed.
Next-generation sequencing (NGS) provides a novel method to uncover transcriptomics data [45, 46]. This technology shows major advantages in robustness, resolution and inter-lab portability over several microarray platforms . These NGS platforms can detect millions of transcripts and can be used for new gene discovery and expression profiling independent of a reference genome [48–50]. In this study, in order to construct a comprehensive transcriptome and investigate the molecular mechanism behind the starch accumulation in L. punctata 0202, samples collected at 0 h, 2 h and 24 time points respectively after fronds were shifted from Hoagland nutrient solution to distilled water were used for a high throughput RNA-Seq analysis. Paired-end (PE) reads from the RNA-Seq were then de novo assembled to build a duckweed transcriptome, which was further used for comparative analysis to reveal the expression patterns of this gene set. This analysis gives a preliminary but global insight into the possible molecular mechanism of starch accumulation, and provides large amount of information for future basic research in duckweed.