Metabolic engineering of sugarcane to accumulate energy‐dense triacylglycerols in vegetative biomass

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy‐rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon‐partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co‐expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1‐2 (DGAT1‐2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP‐glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95‐ or 43‐fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5‐ to 9.5‐fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.     

Engineering triacylglycerol accumulation in duckweed (Lemna japonica)

Duckweeds are amongst the fastest growing of higher plants, making them attractive high-biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol-inducible cyan fluorescent protein-Arabidopsis WRINKLED1 fusion protein (CFP-AtWRI1), strong constitutive expression of a mouse diacylglycerol:acyl-CoA acyltransferase2 (MmDGAT), and a sesame oleosin variant (SiOLE(*)). Individual expression of each gene increased TAG accumulation by 1- to 7-fold relative to controls, while expression of pairs of these genes increased TAG by 7- to 45-fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45-fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108-fold to 8.7% of DW after incubation on medium containing 100 μm estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three-fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY-stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven-fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.     

Survey of duckweed diversity in Lake Chao and total fatty acid,triacylglycerol, profiles of representative strains

Lemnaceae (duckweeds) are widely distributed aquatic flowering plants. Their high growth rate, starch content and suitability for bioremediation make them potential feedstock for biofuels. However, few natural duckweed resources have been investigated in China, and there is no information about total fatty acid (TFA) and triacylglycerol (TAG) composition of duckweeds from China. Here, the genetic diversity of a natural duckweed population collected from Lake Chao, China, was investigated using multilocus sequence typing (MLST). The 54 strains were categorised into four species in four genera, representing 12 distinct sequence types. Strains representing Lemna aequinoctialis and Spirodela polyrhiza were predominant. Interestingly, a surprisingly high degree of genetic diversification within L. aequinoctialis was observed. The four duckweed species revealed a uniform fatty acid composition, with three fatty acids, palmitic acid, linoleic acid and linolenic acid, accounting for more than 80% of the TFA. The TFA in biomass varied among species, ranging from 1.05% (of dry weight, DW) for L. punctata and S. polyrhiza to 1.62% for Wolffia globosa. The four duckweed species contained similar TAG contents, 0.02% mg·DW^?1. The fatty acid profiles of TAG were different from those of TFA, and also varied among the four species. The survey investigated the genetic diversity of duckweeds from Lake Chao, and provides an initial insight into TFA and TAG of four duckweed species, indicating that intraspecific and interspecific variations exist in the content and composition of both TFA and TAG in comparison with other studies.     

Benzyl Amino Purine and Gibberellic Acid Coupled to Nitrogen-Limited Stress Induce Fatty Acids,Biomass Accumulation,and Gene Expression in <i>Scenedesmus Obliquus</i>

The need for renewable energy sources makes microalgae an essential feedstock for biofuels production. The molecular aspects and the response to nitrogen (N)-limited conditions with a phytohormone stimulus in microalgae have been slightly explored. In this work, Scenedesmus obliquus was used as a study model to analyze the effect of benzyl amino purine (BAP) and gibberellic acid (GA) coupled to nitrogen limitation on cell growth, biomass and fatty acids. The selected 10^-5 M BAP increased the biomass by 1.44-fold, and 10^-6 M GA by 1.35-fold. The total lipids also increased by 2.8 and 1.11-fold, respectively. The 10^-5 M BAP and
10^-6 M GA addition to S. obliquus cultures at different initial nitrogen percentages (N-0, N-25, and N-50) showed a significant increase in cell growth and biomass productivity compared to the unstimulated cultures. BAP N-0 and GA N-0 produced the highest lipid yields with 55% and 50%, respectively. The lipid profile analysis revealed an increase, particularly in C18:1 and C16:0 fatty acids. Gene expression analysis showed an over-expression of acyl carrying protein (ACP), stearoyl-ACP desaturase (SAD), fatty acid acyl-ACP thioesterase (FATA), and diacylglycerol acyltransferase (DGAT) genes, which were mainly induced by nitrogen limitation. Furthermore, BAP and GA produced a significant over-expression on these genes in the N-replete cultures. This study shows that BAP and GA, coupled to N limitation stress, can be used to increase the biomass and lipid production in S. obliquus for sustainable biofuels.     

Metabolic engineering of the oleaginous alga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerol by combined pulling and pushing strategies

The marine alga Nannochloropsis oceanica has been considered as a promising photosynthetic cell factory for synthesizing eicosapentaenoic acid (EPA), yet the accumulation of EPA in triacylglycerol (TAG) is restricted to an extreme low level. Poor channeling of EPA to TAG was observed in N. oceanica under TAG induction conditions, likely due to the weak activity of endogenous diacylglycerol acyltransferases (DGATs) on EPA-CoA. Screening over thirty algal DGATs revealed potent enzymes acting on EPA-CoA. Whilst overexpressing endogenous DGATs had no or slight effect on EPA abundance in TAG, introducing selected DGATs with strong activity on EPA-CoA, particularly the Chlamydomonas-derived CrDGTT1, which resided at the outermost membrane of the chloroplast and provided a strong pulling power to divert EPA to TAG for storage and protection, led to drastic increases in EPA abundance in TAG and TAG-derived EPA level in N. oceanica. They were further promoted by additional overexpression of an elongase gene involved in EPA biosynthesis, reaching 5.9- and 12.3-fold greater than the control strain, respectively. Our results together demonstrate the concept of applying combined pulling and pushing strategies to enrich EPA in algal TAG and provide clues for the enrichment of other desired fatty acids in TAG as well.     

Metabolic engineering of biomass for high energy density: oilseed‐like triacylglycerol yields from plant leaves

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co‐expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild‐type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil‐processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.     

Forest Structure and Biomass of a Tropical Seasonal Rain Forest in Xishuangbanna,Southwest China1

Xishuangbanna is a region located at the northern edge of tropical Asia. Biomass estimates of its tropical rain forest have not been published in English literature. We estimated forest biomass and its allocation patterns in five 0.185–1.0 ha plots in tropical seasonal rain forests of Xishuangbanna. Forest biomass ranged from 362.1 to 692.6 Mg/ha. Biomass of trees with diameter at 1.3 m breast height (DBH) ≥ 5 cm accounted for 98.2 percent of the rain forest biomass, followed by shrubs (0.9%), woody lianas (0.8%), and herbs (0.2%). Biomass allocation to different tree components was 68.4–70.0 percent to stems, 19.8–21.8 percent to roots, 7.4–10.6 percent to branches, and 0.7–1.3 percent to leaves. Biomass allocation to the tree sublayers was 55.3–62.2 percent to the A layer (upper layer), 30.6–37.1 percent to the B layer (middle), and 2.7–7.6 percent to the C layer (lower). Biomass of Pometia tomentosa, a dominant species, accounted for 19.7–21.1 percent of the total tree biomass. The average density of large trees (DBH ≥100 cm) was 9.4 stems/ha on two small plots and 3.5 stems/ha on two large plots, illustrating the potential to overestimate biomass on a landscape scale if only small plots are sampled. Biomass estimations are similar to typical tropical rain forests in Southeast Asia and the Neotropics.     

The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp

We examined responses of batch cultures of the marine microalga Nannochloropsis sp. to combined alterations in salinity (13, 27, and 40 g/l NaCl) and light intensity (170 and 700 μmol photons/m²·s). Major growth parameters and lipid productivity (based on total fatty acid determination) were determined in nitrogen-replete and nitrogen-depleted cultures of an initial biomass of 0.8 and 1.4 g/l, respectively. On the nitrogen-replete medium, increases in light intensity and salinity increased the cellular content of dry weight and lipids due to enhanced formation of triacylglycerols (TAG). Maximum average productivity of ca. 410 mg TFA/l/d were obtained at 700 μmol photons/m²·s and 40 g/l NaCl within 7 days. Under stressful conditions, content of the major LC-PUFA, eicosapentaenoic acid (EPA), was significantly reduced while TAG reached 25% of biomass. In contrast, lower salinity tended to improve major growth parameters, consistent with less variation in EPA contents. Combined higher salinity and light intensity was detrimental to lipid productivity under nitrogen starvation; biomass TFA content, and lipid productivity amounted for only 33% of DW and ca. 200 mg TFA/l/day, respectively. The highest biomass TFA content (ca. 47% DW) and average lipid productivity of ca. 360 mg TFA/l/day were achieved at 13 g/l NaCl and 700 μmol photons/m²·s. Our data further support selecting Nannochloropsis as promising microalgae for biodiesel production. Moreover, appropriate cultivation regimes may render Nannochloropsis microalgae to produce simultaneously major valuable components, EPA, and TAG, while sustaining relatively high biomass growth rates.     

Enhanced accumulation of fatty acids and triacylglycerols in transgenic tobacco stems for enhanced bioenergy production

Key message

We report a novel approach for enhanced accumulation of fatty acids and triacylglycerols for utilization as biodiesel in transgenic tobacco stems through xylem-specific expression of Arabidopsis DGAT1 and LEC2 genes.

Abstract

The use of plant biomass for production of bioethanol and biodiesel has an enormous potential to revolutionize the global bioenergy outlook. Several studies have recently been initiated to genetically engineer oil production in seeds of crop plants to improve biodiesel production. However, the “food versus fuel” issues have also sparked some studies for enhanced accumulation of oils in vegetative tissues like leaves. But in the case of bioenergy crops, use of woody stems is more practical than leaves. Here, we report the enhanced accumulation of fatty acids (FAs) and triacylglycerols (TAGs) in stems of transgenic tobacco plants expressing Arabidopsis diacylglycerol acyltransferase 1 (DGAT1) and LEAFY COTYLEDON2 (LEC2) genes under a developing xylem-specific cellulose synthase promoter from aspen trees. The transgenic tobacco plants accumulated significantly higher amounts of FAs in their stems. On an average, DGAT1 and LEC2 overexpression showed a 63 and 80 % increase in total FA production in mature stems of transgenic plants over that of controls, respectively. In addition, selected DGAT1 and LEC2 overexpression lines showed enhanced levels of TAGs in stems with higher accumulation of 16:0, 18:2 and 18:3 TAGs. In LEC2 lines, the relative mRNA levels of the downstream genes encoding plastidic proteins involved in FA synthesis and accumulation were also elevated. Thus, here, we provide a proof of concept for our approach of enhancing total energy yield per plant through accumulation of higher levels of FAs in transgenic stems for biodiesel production.     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

Drought and warming induced changes in P and K concentration and accumulation in plant biomass and soil in a Mediterranean shrubland

A field experiment involving drought and warming manipulation was conducted over a 6-year period in a Mediterranean shrubland to simulate the climate conditions projected by IPCC models for the coming decades (20% decreased soil moisture and 1°C warming). We investigated P and K concentration and accumulation in the leaves and stems of the dominant species, and in soil. Drought decreased P concentration in Globularia alypum leaves (21%) and in Erica multiflora stems (30%) and decreased K concentration in the leaves of both species (20% and 29%, respectively). The general decrease of P and K concentration in drought plots was due to the reduction of soil water content, soil and root phosphatase activity and photosynthetic capacity that decreased plant uptake capacity. Warming increased P concentration in Erica multiflora leaves (42%), but decreased it in the stems and leaf litter of Erica multiflora and the leaf litter (33%) of Globularia alypum, thereby demonstrating that warming improved the P retranslocation and allocation from stem to leaves. These results correlate with the increase in photosynthetic capacity and growth of these two dominant shrub species in warming plots. Drought and warming had no significant effects on biomass P accumulation in the period 1999–2005, but drought increased K accumulation in aboveground biomass (10 kg ha⁻¹) in Globularia alypum due to the increase in K concentration in stems. The stoichiometric changes produced by the different responses of the nutrients led to changes in the P/K concentration ratio in Erica multiflora leaves, stems and litter, and in Globularia alypum stems and litter. This may have implications for the nutritional value of these plant species and plant–herbivore relationships. The effects of climate change on P and K concentrations and contents in Mediterranean ecosystems will differ depending on whether the main component of change is drought or warming.     

Functional characterization of two diacylglycerol acyltransferase 1 genes in Mortierella alpina

Diacylglycerol acyltransferase (DGAT) is a crucial enzyme in the triacylglycerol (TAG) biosynthesis pathway. The oleaginous fungus Mortierella alpina can accumulate large amounts of arachidonic acid (ARA, C20:4) in the form of TAG. Therefore, it is important to study the functional characteristics of its DGAT. Two putative genes MaDGAT1A/1B encoding DGAT1 were identified in M. alpina ATCC 32222 genome by sequence alignment. Sequence alignment with identified DGAT1 homologs showed that MaDGAT1A/1B contain seven conserved motifs that are characteristic of the DGAT1 subfamily. Conserved domain analysis showed that both MaDGAT1A and MaDGAT1B belong to the Membrane-bound O-acyltransferases superfamily. The transforming with MaDGAT1A/1B genes could increase the accumulation of TAG in Saccharomyces cerevisiae to 4·47 and 7·48% of dry cell weight, which was 7·3-fold and 12·3-fold of the control group, respectively, but has no effect on the proportion of fatty acids in TAG. This study showed that MaDGAT1A/1B could effectively promote the accumulation of TAG and therefore may be used in metabolic engineering aimed to increase TAG production of oleaginous fungi.     

Production and nitrogen responses of the African dwarf shrub Indigofera spinosa to defoliation and water limitation

Summary The dwarf shrub Indigofera spinosa Forsk. (Papilionacea), a native forage species of arid Northwest Kenya, was propogated from seed, grown in a controlled environment, and subjected to three treatments of defoliation and watering frequencies in a factorial experimental design. Biomass production and nitrogen accumulation in tissue components were measured to determine defoliation responses in a water-limited environment. We hypothesized that plants would maintain biomass and nitrogen flows despite removal of aboveground meristems and tissues by defoliation. Principal experimental results included a slight reduction (11%; P=0.08) of total biomass production by clipping ca. 1/3 or 2/3 of new leaves and stems and all apical meristems every month. Total aboveground production was not affected by clipping, while final root biomass was reduced 17% by the 2/3 clipping. The least water stressed plants were affected most negatively by defoliation, and the unclipped plants responded more negatively to greater water limitation. Plants achieved partial biomass compensation through alterations in shoot activity and continued allocation of photosynthate to roots. A smaller fraction of leaf production was directed to litter in clipped plants although clipping only removed the youngest tissues, suggesting that clipping increased leaf longevity. In turn, each leaf probably contributed a greater total quantity of photosynthate. Photosynthetic rates were also likely to have been increased by clipping water-stressed plants. In contrast to biomass, plants overcompensated for nitrogen lost to defoliation. Total nitrogen uptake by individual plants was stimulated by defoliation, as there was more total nitrogen in leaves and stems. Increased nitrogen uptake was achieved by clipping stimulation of total uptake per unit of root rather than of total root mass.