Novel resources to investigate leaf plasmodesmata formation in C and C monocots.

Plasmodesmata (PD) are nanochannels that facilitate cell-to-cell transport in plants. More productive and photosynthetically efficient C plants form more PD at the mesophyll (M)-bundle sheath (BS) interface in their leaves than their less efficient C relatives. In C leaves, PD play an essential role in facilitating the rapid metabolite exchange between the M and BS cells to operate a biochemical CO concentrating mechanism, which increases the CO partial pressure at the site of Rubisco in the BS cells and hence photosynthetic efficiency.

The role of SWEET4 proteins in the post-phloem sugar transport pathway of Setaria viridis sink tissues.

In the developing seeds of all higher plants, filial cells are symplastically isolated from the maternal tissue supplying photosynthate to the reproductive structure. Photoassimilates must be transported apoplastically, crossing several membrane barriers, a process facilitated by sugar transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been proposed to play a crucial role in apoplastic sugar transport during phloem unloading and the post-phloem pathway in sink tissues.

A single promoter-TALE system for tissue-specific and tuneable expression of multiple genes in rice.

In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs).

Measuring Plasmodesmata Density on Cell Interfaces of Monocot Leaves Using 3D Immunolocalization and Scanning Electron Microscopy.

Quantification of plasmodesmata density on cell interfaces of plant tissues, particularly of leaves, has been a long-standing challenge. Using electron microscopy alone to quantify plasmodesmata is difficult because of the limited surface area coverage per image and hence the need to examine large numbers of sections for robust quantification. Fluorescence microscopy provides the larger surface area coverage per image but can only visualize pit fields and not individual plasmodesma.

Dark respiration rates are not determined by differences in mitochondrial capacity, abundance and ultrastructure in C leaves.

Our understanding of the regulation of respiration in C plants, where mitochondria play different roles in the different types of C photosynthetic pathway, remains limited. We examined how leaf dark respiration rates (R ), in the presence and absence of added malate, vary in monocots representing the three classical biochemical types of C photosynthesis (NADP-ME, NAD-ME and PCK) using intact leaves and extracted bundle sheath strands. In particular, we explored to what extent rates of R are associated with mitochondrial number, volume and ultrastructure.

Bundle sheath suberisation is required for C photosynthesis in a Setaria viridis mutant.

C photosynthesis provides an effective solution for overcoming the catalytic inefficiency of Rubisco. The pathway is characterised by a biochemical CO concentrating mechanism that operates across mesophyll and bundle sheath (BS) cells and relies on a gas tight BS compartment. A screen of a mutant population of Setaria viridis, an NADP-malic enzyme type C monocot, generated using N-nitroso-N-methylurea identified a mutant with an amino acid change in the gene coding region of the ABCG transporter, a step in the suberin synthesis pathway.

CO diffusion in tobacco: a link between mesophyll conductance and leaf anatomy.

The partial pressure of CO at the sites of carboxylation within chloroplasts depends on the conductance to CO diffusion from intercellular airspace to the sites of carboxylation, termed mesophyll conductance ( ). We investigated how varies with leaf age and through a tobacco () canopy by combining gas exchange and carbon isotope measurements using tunable diode laser spectroscopy. We combined these measurements with the anatomical characterization of leaves.

A low CO2-responsive mutant of Setaria viridis reveals that reduced carbonic anhydrase limits C4 photosynthesis.

In C4 species, β-carbonic anhydrase (CA), localized to the cytosol of the mesophyll cells, accelerates the interconversion of CO2 to HCO3-, the substrate used by phosphoenolpyruvate carboxylase (PEPC) in the first step of C4 photosynthesis. Here we describe the identification and characterization of low CO2-responsive mutant 1 (lcr1) isolated from an N-nitroso-N-methylurea- (NMU) treated Setaria viridis mutant population. Forward genetic investigation revealed that the mutated gene Sevir.

Installation of C photosynthetic pathway enzymes in rice using a single construct.

Introduction of a C photosynthetic mechanism into C crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters.

On the road to C rice: advances and perspectives.

The international C rice consortium aims to introduce into rice a high capacity photosynthetic mechanism, the C pathway, to increase yield. The C pathway is characterised by a complex combination of biochemical and anatomical specialisation that ensures high CO partial pressure at RuBisCO sites in bundle sheath (BS) cells. Here we report an update of the progress of the C rice project.

Response of plasmodesmata formation in leaves of C grasses to growth irradiance.

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C leaves is a key requirement for C photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C species using our PD quantification method, which combines three-dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP-ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 μmol m  s ), and high light (1,000 μmol m  s ), was observed both at seedling and established growth stages.

Diffusion of CO across the Mesophyll-Bundle Sheath Cell Interface in a C Plant with Genetically Reduced PEP Carboxylase Activity.

Phosphopyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C photosynthetic pathway. Here, we used RNA interference to target the cytosolic photosynthetic PEPC isoform in and isolated independent transformants with very low PEPC activities. These plants required high ambient CO concentrations for growth, consistent with the essential role of PEPC in C photosynthesis.

Peeking at a plant through the holes in the wall - exploring the roles of plasmodesmata.

Plasmodesmata (PD) are membrane-lined pores that connect neighbouring plant cells and allow molecular exchange via the symplast. Past studies have revealed the basic structure of PD, some of the transport mechanisms for molecules through PD, and a variety of physiological processes in which they function. Recently, with the help of newly developed technologies, several exciting new features of PD have been revealed.

Multiple mechanisms for enhanced plasmodesmata density in disparate subtypes of C4 grasses.

Proliferation of plasmodesmata (PD) connections between bundle sheath (BS) and mesophyll (M) cells has been proposed as a key step in the evolution of two-cell C4 photosynthesis; However, a lack of quantitative data has hampered further exploration and validation of this hypothesis. In this study, we quantified leaf anatomical traits associated with metabolite transport in 18 species of BEP and PACMAD grasses encompassing four origins of C4 photosynthesis and all three C4 subtypes (NADP-ME, NAD-ME, and PCK). We demonstrate that C4 leaves have greater PD density between M and BS cells than C3 leaves.

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

Targeted Knockdown of GDCH in Rice Leads to a Photorespiratory-Deficient Phenotype Useful as a Building Block for C4 Rice.

The glycine decarboxylase complex (GDC) plays a critical role in the photorespiratory C2 cycle of C3 species by recovering carbon following the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase. Loss of GDC from mesophyll cells (MCs) is considered a key early step in the evolution of C4 photosynthesis. To assess the impact of preferentially reducing GDC in rice MCs, we decreased the abundance of OsGDCH (Os10g37180) using an artificial microRNA (amiRNA) driven by a promoter that preferentially drives expression in MCs.

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.

Strategies for engineering a two-celled C(4) 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.