A short sequence motif that regulates mesophyll-specific accumulation of multiple enzymes is used in C4 photosynthesis.

If the highly efficient C ₄ photosynthesis pathway could be transferred to crops with the C ₃ pathway there could be yield gains of up to 50%. It has been proposed that the multiple metabolic and developmental modifications associated with C ₄ photosynthesis are underpinned by relatively few master regulators that have allowed the evolution of C ₄ photosynthesis more than 60 times in flowering plants. Here we identify a component of one such regulator that consists of a pair of cis -elements located in coding sequence of multiple genes that are preferentially expressed in bundle sheath cells of C ₄ leaves. These motifs represent duons as they play a dual role in coding for amino acids as well as controlling the spatial patterning of gene expression associated with the C ₄ leaf. They act to repress transcription of C ₄ photosynthesis genes in mesophyll cells. These duons are also present in the C ₃ model Arabidopsis thaliana , and, in fact, are conserved in all land plants and even some algae that use C ₃ photosynthesis. C ₄ photosynthesis therefore appears to have coopted an ancient regulatory code to generate the spatial patterning of gene expression that is a hallmark of C ₄ photosynthesis. This intragenic transcriptional regulatory sequence could be exploited in the engineering of efficient photosynthesis of crops.

The majority of plants use C₃ photosynthesis, but over 60 independent lineages of angiosperms have evolved the C₄ pathway. In most C₄ species, photosynthesis gene expression is compartmented between mesophyll and bundle-sheath cells. We performed DNaseI sequencing to identify genome-wide profiles of transcription factor binding in leaves of the C₄ grasses Zea mays, Sorghum bicolor, and Setaria italica as well as C₃Brachypodium distachyon. In C₄ species, while bundle-sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that influence gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C₄ photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C₄ photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general.

When exposed to high light, plants produce reactive oxygen species (ROS). In Arabidopsis thaliana, local stress such as excess heat or light initiates a systemic ROS wave in phloem and xylem cells dependent on NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins. In the case of excess light, although the initial local accumulation of ROS preferentially takes place in bundle-sheath strands, little is known about how this response takes place. Using rice and the ROS probes diaminobenzidine and 2′,7′-dichlorodihydrofluorescein diacetate, we found that, after exposure to high light, ROS were produced more rapidly in bundle-sheath strands than mesophyll cells. This response was not affected either by CO₂ supply or photorespiration. Consistent with these findings, deep sequencing of messenger RNA (mRNA) isolated from mesophyll or bundle-sheath strands indicated balanced accumulation of transcripts encoding all major components of the photosynthetic apparatus. However, transcripts encoding several isoforms of the superoxide/H₂O₂-producing enzyme NADPH oxidase were more abundant in bundle-sheath strands than mesophyll cells. ROS production in bundle-sheath strands was decreased in mutant alleles of the bundle-sheath strand preferential isoform of OsRBOHA and increased when it was overexpressed. Despite the plethora of pathways able to generate ROS in response to excess light, NADPH oxidase–mediated accumulation of ROS in the rice bundle-sheath strand was detected in etiolated leaves lacking chlorophyll. We conclude that photosynthesis is not necessary for the local ROS response to high light but is in part mediated by NADPH oxidase activity. Significance The ability of plants to initiate systemic responses to local biotic and abiotic stress is well known. One such example takes place after exposure to high-light episodes and involves an initial, local accumulation of reactive oxygen species (ROS) in the region exposed to excessive light. Overexcitation of the photosynthetic apparatus has long been known to lead to ROS production, but whether this is necessary and sufficient is not known. Here, we show that local synthesis of ROS also requires NADPH oxidases located in bundle-sheath strands and is not dependent on photosynthesis. As NADPH oxidases also allow the systemic ROS response, this work identifies a component that unifies both the local and systemic responses of plants to high light.

Protoplasts, which are plant cells with their cell walls removed, have been used for decades in plant research and have been instrumental in genetic transformation and the study of various aspects of plant physiology and genetics. With the advent of synthetic biology, these individualized plant cells are fundamental to accelerate the ‘design–build–test–learn’ cycle, which is relatively slow in plant research. Despite their potential, challenges remain in expanding the use of protoplasts in synthetic biology. The capacity of individual protoplasts to hybridize to form new varieties, and to regenerate from single cells, creating individuals with new features is underexplored. The main objective of this review is to discuss the use of protoplasts in plant synthetic biology and to highlight the challenges to exploiting protoplast technologies in this new ‘age of synthetic biology’.