Challenges and Approaches to Crop Improvement Through C3-to-C4 Engineering

With a rapidly growing world population and dwindling natural resources, we are now facing the enormous challenge of increasing crop yields while simultaneously improving the efficiency of resource utilization. Introduction of C4 photosynthesis into C3 crops is widely accepted as a key strategy to meet this challenge because C4 plants are more efficient than C3 plants in photosynthesis and resource usage, particularly in hot climates, where the potential for productivity is high. Lending support to the feasibility of this C3-to-C4 engineering, evidence indicates that C4 photosynthesis has evolved from C3 photosynthesis in multiple lineages. Nevertheless, C3-to-C4 engineering is not an easy task, as several features essential to C4 photosynthesis must be introduced into C3 plants. One such feature is the spatial separation of the two phases of photosynthesis (CO2 fixation and carbohydrate synthesis) into the mesophyll and bundle sheath cells, respectively. Another feature is the Kranz anatomy, characterized by a close association between the mesophyll and bundle sheath (BS) cells (1:1 ratio). These anatomical features, along with a C4-specific carbon fixation enzyme (PEPC), form a CO2-concentration mechanism that ensures a high photosynthetic efficiency. Much effort has been taken in the past to introduce the C4 mechanism into C3 plants, but none of these attempts has met with success, which is in my opinion due to a lack of system-level understanding and manipulation of the C3 and C4 pathways. As a prerequisite for the C3-to-C4 engineering, I propose that not only the mechanisms that control the Kranz anatomy and cell-type-specific expression in C3 and C4 plants must be elucidated, but also a good understanding of the gene regulatory network underlying C3 and C4 photosynthesis must be achieved. In this review, I first describe the past and current efforts to increase photosynthetic efficiency in C3 plants and their limitations; I then discuss a systems approach to tackling down this challenge, some practical issues, and recent technical innovations that would help us to solve these problems.

Transition From Proto-Kranz-Type Photosynthesis to HCO3– Use Photosynthesis in the Amphibious Plant Hygrophila polysperma

Hygrophila polysperma is a heterophyllous amphibious plant. The growth of H. polysperma in submerged conditions is challenging due to the low CO2 environment, increased resistance to gas diffusion, and bicarbonate ion (HCO3–) being the dominant dissolved inorganic carbon source. The submerged leaves of H. polysperma have significantly higher rates of underwater photosynthesis compared with the terrestrial leaves. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate (DIDS), an anion exchanger protein inhibitor, and ethoxyzolamide (EZ), an inhibitor of internal carbonic anhydrase, repressed underwater photosynthesis by the submerged leaves. These results suggested that H. polysperma acclimates to the submerged condition by using HCO3– for photosynthesis. H. polysperma transports HCO3– into the leaf by a DIDS-sensitive HCO3– transporter and converted to CO2 by carbonic anhydrase. Additionally, proteome analysis revealed that submerged leaves accumulated fewer proteins associated with C4 photosynthesis compared with terrestrial leaves. This finding suggested that H. polysperma is capable of C4 and C3 photosynthesis in the terrestrial and submerged leaves, respectively. The ratio of phosphoenol pyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the submerged leaves was less than that in the terrestrial leaves. Upon anatomical observation, the terrestrial leaves exhibited a phenotype similar to the Kranz anatomy found among C4 plants; however, chloroplasts in the bundle sheath cells were not located adjacent to the vascular bundles, and the typical Kranz anatomy was absent in submerged leaves. These results suggest that H. polysperma performs proto-Kranz type photosynthesis in a terrestrial environment and shifts from a proto-Kranz type in terrestrial leaves to a HCO3– use photosynthesis in the submerged environments.

Accelerated remodeling of the mesophyll-bundle sheath interface in the maize C4 cycle mutant leaves

C4 photosynthesis in the maize leaf involves the exchange of organic acids between mesophyll (M) and the bundle sheath (BS) cells. The transport is mediated by plasmodesmata embedded in the suberized cell wall. We examined the maize Kranz anatomy with a focus on the plasmodesma and cell wall suberization with microscopy methods. In the young leaf zone where M and BS cells had indistinguishable proplastids, plasmodesmata were simple and no suberin was detected. In leaf zones where dimorphic chloroplasts were evident, the plasmodesma acquired sphincter and cytoplasmic sleeves, and suberin was discerned. These modifications were accompanied by a drop in symplastic dye mobility at the M-BS boundary. We compared the kinetics of chloroplast differentiation and the modifications in M-BS connectivity in ppdk and dct2 mutants where C4 cycle is affected. The rate of chloroplast diversification did not alter, but plasmodesma remodeling, symplastic transport inhibition, and cell wall suberization were observed from younger leaf zone in the mutants than in wild type. Our results indicate that inactivation of the C4 genes accelerated the changes in the M-BS interface and the reduced permeability suggests that symplastic transport between M and BS could be gated probably for suppressing erroneous flux of C4 metabolites.

Formation of Proto-Kranz in C3 Rice Induced by Spike-Stalk Injection Method

Introduction of C4 photosynthetic traits into C3 crops is an important strategy for improving photosynthetic capacity and productivity. Here, we report the research results of a variant line of sorghum–rice (SR) plant with big panicle and high spikelet density by introducing sorghum genome DNA into rice by spike-stalk injection. The whole-genome resequencing showed that a few sorghum genes could be integrated into the rice genome. Gene expression was confirmed for two C4 photosynthetic enzymes containing pyruvate, orthophosphate dikinase and phosphoenolpyruvate carboxykinase. Exogenous sorghum DNA integration induced a series of key traits associated with the C4 pathway called “proto-Kranz” anatomy, including leaf thickness, bundle sheath number and size, and chloroplast size in bundle sheath cells. Significantly, transgenic plants exhibited enhanced photosynthetic capacity resulting from both photosynthetic CO2-concentrating effect and improved energy balance, which led to an increase in carbohydrate levels and productivity. Furthermore, such rice plant exhibited delayed leaf senescence. In summary, this study provides a proof for the feasibility of inducing the transition from C3 leaf anatomy to proto-Kranz by spike-stalk injection to achieve efficient photosynthesis and increase productivity.

Morphology, anatomy and leaf ultrastructure of Froelichia tomentosa (Mart.) Moq. (Amaranthaceae) - a critically endangered species in Brazil

In Brazil, Froelichia tomentosa (Mart.) Moq. has records of occurrence in Rio Grande do Sul (RS) and Bahia, however, in the former there are indications that its populations are extinct. In the RS, the records are restricted to the region of sandy- fields. In this region, biodiversity has been threatened by advances in agriculture and forestry that intensified in the sandy patch process. Therefore, this work aimed to describe the morphoanatomy and ultrastructure of the leaf blade in Froelichia tomentosa, seeking to correlate leaf characteristics to the environmental conditions. Individuals from different populations in the sand- fields (Pampa biome) were sampled. Leaf blade analyzes were performed by scanning electron microscopy (SEM), transmission electron microscopy (MET) and optical microscopy (MO). The following anatomical features were verified: epidermis with trichomes and stomata in the adaxial and abaxial surface, compact mesophyll, aquiferous hypodermis, Kranz anatomy, and numerous plastoglobules and peroxisomes. The presence of these characters may be related to the adaptation of this species to environment. In addition, we highlight the necessity to create conservation units in the sand-fields region, in order to preserve species as well as that of the present study.

Corrigendum to: Does the C4 plant Trianthema portulacastrum (Aizoaceae) exhibit weakly expressed crassulacean acid metabolism (CAM)?

We examined whether crassulacean acid metabolism (CAM) is present in Trianthema portulacastrum L. (Aizoaceae), a pantropical, salt-tolerant C4 annual herb with atriplicoid-type Kranz anatomy in leaves but not in stems. The leaves of T. portulacastrum are slightly succulent and the stems are fleshy, similar to some species of Portulaca, the only genus known in which C4 and CAM co-occur. Low- level nocturnal acidification typical of weakly expressed, predominantly constitutive CAM was measured in plants grown for their entire life-cycle in an outdoor raised garden box. Acidification was greater in stems than in leaves. Plants showed net CO2 uptake only during the light irrespective of soil water availability. However, nocturnal traces of CO2 exchange exhibited curved kinetics of reduced CO2 loss during the middle of the night consistent with low-level CAM. Trianthema becomes the second genus of vascular land plants in which C4 and features of CAM have been demonstrated to co-occur in the same plant and the first C4 plant with CAM-type acidification described for the Aizoaceae. Traditionally the stems of herbs are not sampled in screening studies. Small herbs with mildly succulent leaves and fleshy stems might be a numerically significant component of CAM biodiversity.

Anatomy of vegetative organs in Allionia (Nyctaginaceae), with emphasis on the vascular system

Allionia is a small genus within the tribe Nyctagineae (Nyctaginaceae) which has a controversial, infrageneric delimitation. Here, we investigated the two known species of Allionia in order to characterize the anatomy of leaves, stems and roots, with further notes on vascular system development. Additionally, the present study aimed to broaden our knowledge of stem vascular diversity and to survey for anatomical features with diagnostic value in distinguishing A. choisyi from A. incarnata. Leaf anatomy of other Nyctagineae taxa was also analysed. Anatomical and ontogenetic observations from the vegetative organs in Allionia revealed no diagnostic features to distinguish the two species. We illustrated the occurrence of Kranz anatomy, which in Nyctaginaceae is only known in Allionia, Boerhavia, and Okenia. The stem primary vascular system was unusual in showing a polycyclic eustele (medullary bundles + continuous concentric procambium). Likewise, mature stems and roots show vascular cambial variants (successive cambia) that arise from the pericycle. The anatomy and histochemistry of multicellular glan-dular trichomes observed in aerial organs were presented. Raphids were seen in all organs. Although no strong xerophytic features were observed in Allionia, several characteristics can be associated with their arid habitats. Our findings on the vascular system of Allionia showed the two species to be much the same and reinforced earlier findings that the stem anatomy of Nyctaginaceae is complex and intriguing.

Combining the small South American genus Androtrichum into Cyperus (Cyperaceae)

Background and aims – Molecular phylogenetic studies have provided a clearer understanding of the complex relationships within the family Cyperaceae. These studies have consistently shown that 12 allied genera are nested in the genus Cyperus. However, early Sanger sequencing-based phylogenies that included the two species of the small South American genus Androtrichum were inconclusive in placing this genus either as sister to Cyperus or as part of its early divergent lineages. A recent phylogenetic analysis however conclusively placed the two species of Androtrichum within the C3 Cyperus Grade. In this study, we investigate if the morphology and anatomy of Androtrichum species provide additional evidence for their placement in the genus Cyperus. In addition, we provide descriptions and distribution data for the species.Material and methods – Herbarium material from FLOR, FURB, GENT, ICN, and K has been studied. Samples of the culm and leaf were studied using histological methods. Taxonomic changes and typifications of names were performed according to the International Code of Nomenclature for algae, fungi, and plants. Key results – The morphology and the non-Kranz anatomy observed in the Androtrichum species confirm their placement among the other C3 Cyperus species. Androtrichum is combined into Cyperus. For one species, a combination in Cyperus is already available: Cyperus trigynus. For the other species, a new name in Cyperus is published: Cyperus byssaceus. Two typifications are established and morphological descriptions and distribution data are provided. Conclusion – By integrating recent molecular phylogenetic data with additional evidence from morphology and anatomy, Androtrichum is combined into Cyperus. As a result of this taxonomic change, a single monophyletic genus Cyperus is now recognised in the Cyperus Clade of tribe Cypereae.