Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy

The C4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because C4 plants are higher yielding than C3 plants, efforts are underway to introduce the C4 pathway into the C3 crop rice. This is an ambitious endeavor; however, the C4 pathway evolved from C3 on multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering C4 rice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from C3 to so-called “proto-Kranz” anatomy requires an increase in organelle volume in sheath cells surrounding leaf veins. Here we induced chloroplast and mitochondrial development in rice vascular sheath cells through constitutive expression of maize GOLDEN2-LIKE genes. Increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased intercellular connections. This suite of traits reflects that seen in “proto-Kranz” species, and, as such, a key step toward engineering C4 rice has been achieved.Research was funded by a C4 Rice Project grant from The Bill & Melinda Gates Foundation to IRRI (2012–2015; OPPGD1394) and the University of Oxford (2015–2019; OPP1129902)

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

Summary: 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

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter disturbs carbon and nitrogen homeostasis in rice

The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of OAA. Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT (glutamine synthetase/glutamate synthase) reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis: OAA resulting from phosphoenolpyruvate carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-malate dehydrogenase, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. To engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in transgenic rice was confirmed by reconstitution of transporter activity into proteoliposomes. Unexpectedly, overexpression of ZmOMT1 in rice negatively affected growth, CO2 assimilation rate, total free amino acid content, tricarboxylic acid cycle metabolites, as well as sucrose and starch contents. Accumulation of high amounts of aspartate and the impaired growth phenotype of OMT1 rice lines could be suppressed by simultaneous overexpression of ZmDiT2. Implications for engineering C4 rice are discussed

The relationship and different C4 Kranz anatomy of Bassia eriantha and Bassia eriophora, two often confused Irano-Turanian and Saharo-Sindian species

Akhani, Hossein, Khoshravesh, Roxana (2013): The relationship and different C Kranz anatomy of Bassia eriantha and Bassia eriophora, two often confused Irano-Turanian and Saharo-Sindian species. Phytotaxa 93 (1): 1-24, DOI: 10.11646/phytotaxa.93.1.1, URL: http://dx.doi.org/10.11646/phytotaxa.93.1.

Leaf Microscopy Applications in Photosynthesis Research:Identifying the Gaps

Leaf imaging via microscopy has provided critical insights into research on photosynthesis at multiple junctures, from the early understanding of the role of stomata, through elucidating C4 photosynthesis via Kranz anatomy and chloroplast arrangement in single cells, to detailed explorations of diffusion pathways and light utilization gradients within leaves. In recent decades, the original two-dimensional (2D) explorations have begun to be visualized in three-dimensional (3D) space, revising our understanding of structure-function relationships between internal leaf anatomy and photosynthesis. In particular, advancing new technologies and analyses are providing fresh insight into the relationship between leaf cellular components and improving the ability to model net carbon fixation, water use efficiency, and metabolite turnover rate in leaves. While ground-breaking developments in imaging tools and techniques have expanded our knowledge of leaf 3D structure via high-resolution 3D and time-series images, there is a growing need for more in vivo imaging as well as metabolite imaging. However, these advances necessitate further improvement in microscopy sciences to overcome the unique challenges a green leaf poses. In this review, we discuss the available tools, techniques, challenges, and gaps for efficient in vivo leaf 3D imaging, as well as innovations to overcome these difficulties

A review of plant diversity, vegetation, and phytogeography of the Khorassan-Kopet Dagh floristic province in the Irano-Turanian region (northeastern Iran–southern Turkmenistan)

The Khorassan-Kopet Dagh (KK) floristic province is located in the northeastern parts of Iran and partly in southern Turkmenistan. The area is a transition zone and a corridor connecting different provinces of the Irano-Turanian region and also Hyrcanian montane forests of the Euro-Siberian region. The unique combination of Irano-Turanian species and also presence of a local center of endemism are evidence of a separate biogeographic entity. The complicated topography, high habitat heterogeneity and vegetation history are reasons for the development of diverse vegetation types. In order to achieve up-to-date information on the plant diversity and distribution patterns, a database was prepared using all floristic records from the area defined as KK. A total of 2576 species/infraspecific taxa belonging to 702 genera and 112 families of vascular plants have been reported from the area, 2498 of which occur within Iran. Altogether, 28 different distribution patterns are recognized among five major phytogeographical groups, including widespread, tri-regional, bi-regional, Euro-Siberian and Irano-Turanian patterns. Irano-Turanian elements, which make up the core flora of KK, are subdivided further into 14 distribution patterns. A significant number of species, i.e. 356 species (13.8%), are endemic to the area. The flora of KK is highly influenced by central Irano-Turanian elements. The main vegetation types of the area include Juniperus woodlands, Pistacia vera woodlands, some isolated enclaves of Hyrcanian forests and scrub, cliff vegetation, mountain steppe communities, semi-desert steppes, loess and marl vegetation, halophytic vegetation, aquatic and hygrophilous communities, and ruderal/invasive plant communities. There are several major threats to the ecosystems and biodiversity of the area. The areas presently protected do not cover all of the vegetation types, and therefore many threatened species are not safe.</jats:p

From proto-Kranz to C4 Kranz: building the bridge to C4 photosynthesis

In this review, we examine how the specialized “Kranz” anatomy of C4 photosynthesis evolved from C3 ancestors. Kranz anatomy refers to the wreath-like structural traits that compartmentalize the biochemistry of C4 photosynthesis and enables the concentration of CO2 around Rubisco. A simplified version of Kranz anatomy is also present in the species that utilize C2 photosynthesis, where a photorespiratory glycine shuttle concentrates CO2 into an inner bundle-sheath-like compartment surrounding the vascular tissue. C2 Kranz is considered to be an intermediate stage in the evolutionary development of C4 Kranz, based on the intermediate branching position of C2 species in 14 evolutionary lineages of C4 photosynthesis. In the best-supported model of C4 evolution, Kranz anatomy in C2 species evolved from C3 ancestors with enlarged bundle sheath cells and high vein density. Four independent lineages have been identified where C3 sister species of C2 plants exhibit an increase in organelle numbers in the bundle sheath and enlarged bundle sheath cells. Notably, in all of these species, there is a pronounced shift of mitochondria to the inner bundle sheath wall, forming an incipient version of the C2 type of Kranz anatomy. This incipient version of C2 Kranz anatomy is termed proto-Kranz, and is proposed to scavenge photorespiratory CO2. By doing so, it may provide fitness benefits in hot environments, and thus represent a critical first stage of the evolution of both the C2 and C4 forms of Kranz anatomy

A new species ofBienertia(Chenopodiaceae) from Iranian salt deserts:A third species of the genus and discovery of a fourth terrestrial C4plant without Kranz anatomy

Bienertia is a very interesting genus with its unique C4 photosynthesis in a single cell. Recent investigations on the taxonomy of the genus using a multidisciplinary approach revealed the existence a third species of this genus from the margin of Dasht-e Kavir (desert plain) in central Iran, thus adding a fourth terrestrial C4 plant lacking Kranz anatomy. The flattened leaves, the semi-inferior ovary resulting from adnation of the perianth with the ovary, in addition to cotyledon morphology and hypocotyl length, provide evidence for the existence of a new species. The new species is here described as Bienertia kavirense Akhani spec. nov., after its locality at the margin of the Kavir. The gametic chromosome complement of the new species is n = 9. The carbon isotope values (δ13C) showed a C4 photosynthesis which is remarkably less negative than in the two other species of Bienertia. Detailed information on the morphology, leaf anatomy, and ecology of the new species is provided, and the new association “Bienertio kavirense–Cornulacetum aucheri” is described as a unique plant community occurring at the margin of the Dasht-e Kavir. Only a few species, such as Salsola annua (Bunge) Akhani comb. nov., associate with B. kavirense