William Paul Quick

Strategies for engineering a two-celled C4 photosynthetic pathway into rice

Every day almost one billion people suffer from chronic hunger, and the situation is expected to deteriorate with a projected population growth to 9 billion worldwide by 2050. In order to provide adequate nutrition into the future, rice yields in Asia need to increase by 60%, a change that may be achieved by introduction of the C(4) photosynthetic cycle into rice. The international C(4) Rice Consortium was founded in order to test the feasibility of installing the C(4) engine into rice. This review provides an update on two of the many approaches employed by the C(4) Rice Consortium: namely, metabolic C(4) engineering and identification of determinants of leaf anatomy by mutant screens. The aim of the metabolic C(4) engineering approach is to generate a two-celled C(4) shuttle in rice by expressing the classical enzymes of the NADP-ME C(4) cycle in a cell-appropriate manner. The aim is also to restrict RuBisCO and glycine decarboxylase expression to the bundle sheath (BS) cells of rice in a C(4)-like fashion by specifically down-regulating their expression in rice mesophyll (M) cells. In addition to the changes in biochemistry, two-celled C(4) species show a convergence in leaf anatomy that include increased vein density and reduced numbers of M cells between veins. By screening rice activation-tagged lines and loss-of-function sorghum mutants we endeavour to identify genes controlling these key traits.

Characterization of statistical features for plant microRNA prediction

BackgroundSeveral tools are available to identify miRNAs from deep-sequencing data, however, only a few of them, like miRDeep, can identify novel miRNAs and are also available as a standalone application. Given the difference between plant and animal miRNAs, particularly in terms of distribution of hairpin length and the nature of complementarity with its duplex partner (or miRNA star), the underlying (statistical) features of miRDeep and other tools, using similar features, are likely to get affected.ResultsThe potential effects on features, such as minimum free energy, stability of secondary structures, excision length, etc., were examined, and the parameters of those displaying sizable changes were estimated for plant specific miRNAs. We found most of these features acquired a new set of values or distributions for plant specific miRNAs. While the length of conserved positions (nucleus) in mature miRNAs were relatively longer in plants, the difference in distribution of minimum free energy, between real and background hairpins, was marginal. However, the choice of source (species) of background sequences was found to affect both the minimum free energy and miRNA hairpin stability. The new parameters were tested on an Illumina dataset from maize seedlings, and the results were compared with those obtained using default parameters. The newly parameterized model was found to have much improved specificity and sensitivity over its default counterpart.ConclusionsIn summary, the present study reports behavior of few general and tool-specific statistical features for improving the prediction accuracy of plant miRNAs from deep-sequencing data.

Fine and coarse regulation of reactive oxygen species in the salt tolerant mutants of barnyard grass and their wild-type parents under salt stress

The growth of the wild-type and three salt tolerant mutants of barnyard grass (Echinochloa crusgalli L.) under salt stress was investigated in relation to oxidative stress and activities of the antioxidant enzymes superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), phenol peroxidase (POD: EC 1.11.1.7), glutathione reductase (GR: EC 1.8.1.7) and ascorbate peroxidase (APX: EC 1.11.1.1). The three mutants (fows B17, B19 and B21) grew significantly better than the wild-type under salt stress (200 mM NaCl) but some salt sensitive individuals were still detectable in the populations of the mutants though in smaller numbers compared with the wild-type. The salt sensitive plants had slower growth rates, higher rates of lipid peroxidation and higher levels of reactive oxygen species (ROS) in their leaves compared with the more tolerant plants from the same genotype. These sensitivity responses were maximized when the plants were grown under high light intensity suggesting that the chloroplast could be a main source of ROS under salt stress. However, the salt sensitivity did not correlate with reduced K(+)/Na(+) ratios or enhanced Na(+) uptake indicating that the sensitivity responses may be mainly because of accumulation of ROS rather than ion toxicity. SOD activities did not correlate to salt tolerance. Salt stress resulted in up to 10-fold increase in CAT activity in the sensitive plants but lower activities were found in the tolerant ones. In contrast, the activities of POD, APX and GR were down regulated in the sensitive plants compared with the tolerant ones. A correlation between plant growth, accumulation of ROS and differential modulation of antioxidant enzymes is discussed. We conclude that loss of activities of POD, APX and GR causes loss of fine regulation of ROS levels and hence the plants experience oxidative stress although they have high CAT activities.

C4 Rice – an Ideal Arena for Systems Biology Research

Engineering the C4 photosynthetic pathway into C3 crops has the potential to dramatically increase the yields of major C3 crops. The genetic control of features involved in C4 photosynthesis are still far from being understood; which partially explains why we have gained little success in C4 engineering thus far. Next generation sequencing techniques and other high throughput technologies are offering an unprecedented opportunity to elucidate the developmental and evolutionary processes of C4 photosynthesis. Two contrasting hypotheses about the evolution of C4 photosynthesis exist, i.e. the master switch hypothesis and the incremental gain hypothesis. These two hypotheses demand two different research strategies to proceed in parallel to maximize the success of C4 engineering. In either case, systems biology research will play pivotal roles in identifying key regulatory elements controlling development of C4 features, identifying essential biochemical and anatomical features required to achieve high photosynthetic efficiency, elucidating genetic mechanisms underlining C4 differentiation and ultimately identifying viable routes to engineer C4 rice. As a highly interdisciplinary project, the C4 rice project will have far-reaching impacts on both basic and applied research related to agriculture in the 21st century.

Antisense suppression of the small chloroplast protein CP12 in tobacco alters carbon partitioning and severely restricts growth

The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants.

Improvement of photosynthesis in rice (Oryza sativa L.) by inserting the C4 pathway

To boost food production for a rapidly growing global population, crop yields must significantly increase. One of the avenues being recently explored is the improvement of photosynthetic capacity by installing the C4 photosynthetic pathway into C3 crops like rice to drastically increase their yield. Crops with an enhanced photosynthetic mechanism would better utilize the solar radiation that can be translated into yield. This subsequently will help in producing more grain yield, reduce water loss and increase nitrogen use efficiency especially in hot and dry environments. This review provides a summary of the factors that need to be modified in rice so that the C4 pathway can be introduced successfully. It also discusses the differences between the C3 and C4 photosynthetic pathways in terms of anatomy, biochemistry and genetics.

Two forward genetic screens for vein density mutants in sorghum converge on a cytochrome P450 gene in the brassinosteroid pathway

The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C(4) traits into C(3) plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C(4) plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C(4) species from C(3) species. To identify genetic factors that specify C(4) leaf anatomy, we generated ethyl methanesulfonate- and γ-ray-mutagenized populations of the C(4) species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F(2) populations as homozygous recessive alleles. Bulk segregant analysis using next-generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C(4) and C(3) leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.

Increasing Leaf Vein Density by Mutagenesis: Laying the Foundations for C4 Rice

A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens.

The Metabolite Pathway between Bundle Sheath and Mesophyll: Quantification of Plasmodesmata in Leaves of C3 and C4 Monocots

Scanning electron microscopy and three-dimensional immunolocalization reveal that leaves of C4 monocots have more plasmodesmata per pitfield area and greater pitfield area coverage than C3 monocots. C4 photosynthesis is characterized by a CO2-concentrating mechanism between mesophyll (M) and bundle sheath (BS) cells of leaves. This generates high metabolic fluxes between these cells, through interconnecting plasmodesmata (PD). Quantification of these symplastic fluxes for modeling studies requires accurate quantification of PD, which has proven difficult using transmission electron microscopy. Our new quantitative technique combines scanning electron microscopy and 3D immunolocalization in intact leaf tissues to compare PD density on cell interfaces in leaves of C3 (rice [Oryza sativa] and wheat [Triticum aestivum]) and C4 (maize [Zea mays] and Setaria viridis) monocot species. Scanning electron microscopy quantification of PD density revealed that C4 species had approximately twice the number of PD per pitfield area compared with their C3 counterparts. 3D immunolocalization of callose at pitfields using confocal microscopy showed that pitfield area per M-BS interface area was 5 times greater in C4 species. Thus, the two C4 species had up to nine times more PD per M-BS interface area (S. viridis, 9.3 PD µm−2; maize, 7.5 PD µm−2; rice 1.0 PD µm−2; wheat, 2.6 PD µm−2). Using these anatomical data and measured photosynthetic rates in these C4 species, we have now calculated symplastic C4 acid flux per PD across the M-BS interface. These quantitative data are essential for modeling studies and gene discovery strategies needed to introduce aspects of C4 photosynthesis to C3 crops.

Study of Flowering Pattern in Setaria viridis, a Proposed Model Species for C4 Photosynthesis Research

Green foxtail millet (Setaria viridis) has NADP-ME type of C4 photosynthesis. Because of its short life cycle, small genome size of ~515 Mb, small plant stature, high number of seed set, simple growth requirements, and wide adaptability, this diploid () weed is proposed to be a model species for the study of C4 photosynthesis. It is also a representative of bioenergy grasses and a model for genetic study of invasive weeds. Despite having all traits of a model species, it is difficult to cross-pollinate because its flowering behavior is not well studied. We used time lapse digital recording to study the flowering time and pattern along a single panicle. We found that flowering in Setaria was triggered by the darkness of the night and when the temperature was lower than 35°C. The anthesis of all the spikelets in a panicle took up-to three nights flowering from 9:30 pm to 10:00 am in the morning. Each spikelet has three phases of anthesis during which pollination occurs. A spikelet remains open for less than three hours. The pollination time for each spikelet is less than 60 minutes. Information from this study will facilitate the geneticists and plant breeders to plan for efficient crossing of Setaria.