Engineering chloroplast development in rice through cell-specific control of endogenous genetic circuits.

The engineering of C4 photosynthetic activity into the C3 plant rice has the potential to nearly double rice yields. To engineer a two-cell photosynthetic system in rice, the rice bundle sheath (BS) must be rewired to enhance photosynthetic capacity. Here, we show that BS chloroplast biogenesis is enhanced when the transcriptional activator, Oryza sativa Cytokinin GATA transcription factor 1 (OsCGA1), is driven by a vascular specific promoter. Ectopic expression of OsCGA1 resulted in increased BS chloroplast planar area and increased expression of photosynthesis-associated nuclear genes (PhANG), required for the biogenesis of photosynthetically active chloroplasts in BS cells of rice. A further refinement using a DNAse dead Cas9 (dCas9) activation module driven by the same cell-type specific promoter, directed enhanced chloroplast development of the BS cells when gRNA sequences were delivered by the dCas9 module to the promoter of the endogenous OsCGA1 gene. Single gRNA expression was sufficient to mediate the transactivation of both the endogenous gene and a transgenic GUS reporter fused with OsCGA1 promoter. Our results illustrate the potential for tissue-specific dCas9-activation and the co-regulation of genes needed for multistep engineering of C4 rice.

Biochemical and physiological flexibility accompanies reduced cellulose biosynthesis in Brachypodium cesa1 S830N.

Here, we present a study into the mechanisms of primary cell wall cellulose formation in grasses, using the model cereal grass Brachypodium distachyon. The exon found adjacent to the BdCESA1 glycosyltransferase QXXRW motif was targeted using Targeting Induced Local Lesions in Genomes (TILLING) and sequencing candidate amplicons in multiple parallel reactions (SCAMPRing) leading to the identification of the Bdcesa1 S830N allele. Plants carrying this missense mutation exhibited a significant reduction in crystalline cellulose content in tissues that rely on the primary cell wall for biomechanical support. However, Bdcesa1 S830N plants failed to exhibit the predicted reduction in plant height. In a mechanism unavailable to eudicotyledons, B. distachyon plants homozygous for the Bdcesa1 S830N allele appear to overcome the loss of internode expansion anatomically by increasing the number of nodes along the stem. Stem biomechanics were resultantly compromised in Bdcesa1 S830N . The Bdcesa1 S830N missense mutation did not interfere with BdCESA1 gene expression. However, molecular dynamic simulations of the CELLULOSE SYNTHASE A (CESA) structure with modelled membrane interactions illustrated that Bdcesa1 S830N exhibited structural changes in the translated gene product responsible for reduced cellulose biosynthesis. Molecular dynamic simulations showed that substituting S830N resulted in a stabilizing shift in the flexibility of the class specific region arm of the core catalytic domain of CESA, revealing the importance of this motion to protein function.

Metabolomics of sorghum roots during nitrogen stress reveals compromised metabolic capacity for salicylic acid biosynthesis.

Sorghum (Sorghum bicolor [L.] Moench) is the fifth most productive cereal crop worldwide with some hybrids having high biomass yield traits making it promising for sustainable, economical biofuel production. To maximize biofuel feedstock yields, a more complete understanding of metabolic responses to low nitrogen (N) will be useful for incorporation in crop improvement efforts. In this study, 10 diverse sorghum entries (including inbreds and hybrids) were field-grown under low and full N conditions and roots were sampled at two time points for metabolomics and 16S amplicon sequencing. Roots of plants grown under low N showed altered metabolic profiles at both sampling dates including metabolites important in N storage and synthesis of aromatic amino acids. Complementary investigation of the rhizosphere microbiome revealed dominance by a single operational taxonomic unit (OTU) in an early sampling that was taxonomically assigned to the genus Pseudomonas. Abundance of this Pseudomonas OTU was significantly greater under low N in July and was decreased dramatically in September. Correlation of Pseudomonas abundance with root metabolites revealed a strong negative association with the defense hormone salicylic acid (SA) under full N but not under low N, suggesting reduced defense response. Roots from plants with N stress also contained reduced phenylalanine, a precursor for SA, providing further evidence for compromised metabolic capacity for defense response under low N conditions. Our findings suggest that interactions between biotic and abiotic stresses may affect metabolic capacity for plant defense and need to be concurrently prioritized as breeding programs become established for biofuels production on marginal soils.

Shape and size dependent nonlinear refraction and absorption in citrate-stabilized, near-IR plasmonic silver nanopyramids.

Using a combination of a mild stabilizer and a mild reductant, sodium citrate and hydrazine hydrate, anisotropic silver nanocrystals (NCs) were synthesized with tunable plasmon peaks at 550 nm, 700 nm, 800 nm, 900 nm and 1010 nm (the samples are named Ag-550, Ag-700, Ag-800, Ag-900 and Ag-1010, respectively). TEM investigations revealed that Ag-550 NCs were pentagonal nanoplates while the other four samples were nanopyramids with a pentagonal base with the edge length varying between 15 and 30 nm. The non-linear optical (NLO) properties of these NCs were studied by the Z-scan technique using the CW He-Ne laser (632.8 nm, 15 mW). The shape change from 2D nanoplates (Ag-550) to 3D nanopyramids (Ag-700) resulted in sign reversal of the non-linear refractive index, n2, from a negative (-3.164 × 10-8 cm2 W-1) to a positive one (1.195 × 10-8 cm2 W-1). This corresponds to a change from a self-defocussing effect to a self-focussing one. Besides shape, the size effect is also prominently observed. Amongst nanopyramids, as the edge length increases, n2 increases linearly and reaches a maximum of 3.124 × 10-8 cm2 W-1. Doubling the edge length from 15 nm to 30 nm resulted in 162% increase in n2. On moving from Ag-550 to Ag-900 NCs, with the increasing plasmon wavelength, the non-linear absorption (NLA) coefficient increased exponentially to a high value of 8.52 × 10-4 cm W-1. However, Ag-1010 showed 29% decrease in NLA which is attributed to twinning present in the crystal structure as seen in the HR-TEM images. Due to the tunable NLO properties, these anisotropic Ag NCs hold great potential for applications in optical limiting, switching and data storage.

Evolutionary divergence of phytochrome protein function in Zea mays PIF3 signaling.

Two maize phytochrome-interacting factor (PIF) basic helix-loop-helix (bHLH) family members, ZmPIF3.1 and ZmPIF3.2, were identified, cloned and expressed in vitro to investigate light-signaling interactions. A phylogenetic analysis of sequences of the maize bHLH transcription factor gene family revealed the extent of the PIF family, and a total of seven predicted PIF-encoding genes were identified from genes encoding bHLH family VIIa/b proteins in the maize genome. To investigate the role of maize PIFs in phytochrome signaling, full-length cDNAs for phytochromes PhyA2, PhyB1, PhyB2 and PhyC1 from maize were cloned and expressed in vitro as chromophorylated holophytochromes. We showed that ZmPIF3.1 and ZmPIF3.2 interact specifically with the Pfr form of maize holophytochrome B1 (ZmphyB1), showing no detectable affinity for the Pr form. Maize holophytochrome B2 (ZmphyB2) showed no detectable binding affinity for PIFs in either Pr or Pfr forms, but phyB Pfr from Arabidopsis interacted with ZmPIF3.1 similarly to ZmphyB1 Pfr. We conclude that subfunctionalization at the protein-protein interaction level has altered the role of phyB2 relative to that of phyB1 in maize. Since the phyB2 mutant shows photomorphogenic defects, we conclude that maize phyB2 is an active photoreceptor, without the binding of PIF3 seen in other phyB family proteins.

A Versatile Phenotyping System and Analytics Platform Reveals Diverse Temporal Responses to Water Availability in Setaria.

Phenotyping has become the rate-limiting step in using large-scale genomic data to understand and improve agricultural crops. Here, the Bellwether Phenotyping Platform for controlled-environment plant growth and automated multimodal phenotyping is described. The system has capacity for 1140 plants, which pass daily through stations to record fluorescence, near-infrared, and visible images. Plant Computer Vision (PlantCV) was developed as open-source, hardware platform-independent software for quantitative image analysis. In a 4-week experiment, wild Setaria viridis and domesticated Setaria italica had fundamentally different temporal responses to water availability. While both lines produced similar levels of biomass under limited water conditions, Setaria viridis maintained the same water-use efficiency under water replete conditions, while Setaria italica shifted to less efficient growth. Overall, the Bellwether Phenotyping Platform and PlantCV software detected significant effects of genotype and environment on height, biomass, water-use efficiency, color, plant architecture, and tissue water status traits. All ∼ 79,000 images acquired during the course of the experiment are publicly available.

Evolution and selection of Rhg1, a copy-number variant nematode-resistance locus.

The soybean cyst nematode (SCN) resistance locus Rhg1 is a tandem repeat of a 31.2 kb unit of the soybean genome. Each 31.2-kb unit contains four genes. One allele of Rhg1, Rhg1-b, is responsible for protecting most US soybean production from SCN. Whole-genome sequencing was performed, and PCR assays were developed to investigate allelic variation in sequence and copy number of the Rhg1 locus across a population of soybean germplasm accessions. Four distinct sequences of the 31.2-kb repeat unit were identified, and some Rhg1 alleles carry up to three different types of repeat unit. The total number of copies of the repeat varies from 1 to 10 per haploid genome. Both copy number and sequence of the repeat correlate with the resistance phenotype, and the Rhg1 locus shows strong signatures of selection. Significant linkage disequilibrium in the genome outside the boundaries of the repeat allowed the Rhg1 genotype to be inferred using high-density single nucleotide polymorphism genotyping of 15 996 accessions. Over 860 germplasm accessions were found likely to possess Rhg1 alleles. The regions surrounding the repeat show indications of non-neutral evolution and high genetic variability in populations from different geographic locations, but without evidence of fixation of the resistant genotype. A compelling explanation of these results is that balancing selection is in operation at Rhg1.

Changes of splanchnic perfusion after applying positive end expiratory pressure in patients with acute respiratory distress syndrome.

BACKGROUND: Positive end-expiratory pressure (PEEP) improves oxygenation and can prevent ventilator- induced lung injury in patients with acute respiratory distress syndrome (ARDS). Nevertheless, PEEP can also induce detrimental effects by its influence on the cardiovascular system. The purpose of this study was to assess the effects of PEEP on gastric mucosal perfusion while applying a protective ventilatory strategy in patients with ARDS. MATERIALS AND METHODS: Thirty-two patients were included in the study. A pressure-volume curve was traced and ideal PEEP, defined as lower inflection point + 2cmH(2)O, was determined. Gastric tonometry was measured continuously (Tonocap). After baseline measurements, 10, 15 and 20cmH(2)O PEEP and ideal PEEP were applied for 30 min each. By the end of each period, hemodynamics, CO(2) gap (gastric minus arterial partial pressures), and ventilatory measurements were taken. RESULTS: PEEP had no effect on CO(2) gap (median [range], baseline: 18 [2-30] mmHg; PEEP 10: 18 [0-40] mmHg; PEEP 15: 17 [0-39] mmHg; PEEP 20: 16 [4-39] mmHg; ideal PEEP: 19 [9-39] mmHg; P = 0.19). Cardiac index also remained unchanged (baseline: 4.7 [2.6-6.2] l min(-1) m(-2) ; PEEP 10: 4.4 [2.5-7] l min(-1) m(-2) ; PEEP 15: 4.4 [2.2-6.8] l min(-1) m(-2) ; PEEP 20: 4.8 [2.4-6.3] l min(-1) m(-2) ; ideal PEEP: 4.9 [2.4-6.3] l min(-1) m(-2) ; P = 0.09). CONCLUSION: PEEP of 10-20 cmH(2)O does not affect splanchnic perfusion and is hemodynamically well tolerated in most patients with ARDS, including those receiving inotropic supports.