Introduction of glucan synthase into the cytosol in wheat endosperm causes massive maltose accumulation and represses starch synthesis.

We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic β-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of β-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan

Plastidial NAD-Dependent Malate Dehydrogenase: A Moonlighting Protein Involved in Early Chloroplast Development through Its Interaction with an FtsH12-FtsHi Protease Complex

Malate dehydrogenases (MDHs) convert malate to oxaloacetate using NAD(H) or NADP(H) as a cofactor. mutants lacking plastidial NAD-dependent MDH () are embryo-lethal, and constitutive silencing (1) causes a pale, dwarfed phenotype. The reason for these severe phenotypes is unknown. Here, we rescued the embryo lethality of via embryo-specific expression of pdNAD-MDH. Rescued seedlings developed white leaves with aberrant chloroplasts and failed to reproduce. Inducible silencing of pdNAD-MDH at the rosette stage also resulted in white newly emerging leaves. These data suggest that pdNAD-MDH is important for early plastid development, which is consistent with the reductions in major plastidial galactolipid, carotenoid, and protochlorophyllide levels in 1 seedlings. Surprisingly, the targeting of other NAD-dependent MDH isoforms to the plastid did not complement the embryo lethality of , while expression of enzymatically inactive pdNAD-MDH did. These complemented plants grew indistinguishably from the wild type. Both active and inactive forms of pdNAD-MDH interact with a heteromeric AAA-ATPase complex at the inner membrane of the chloroplast envelope. Silencing the expression of FtsH12, a key member of this complex, resulted in a phenotype that strongly resembles 1. We propose that pdNAD-MDH is essential for chloroplast development due to its moonlighting role in stabilizing FtsH12, distinct from its enzymatic function

Variations in the Calvin-Benson cycle: selection pressures and optimization?

This article comments on: Arrivault S, Moraes TA, Obata T, Medeiros DB, Fernie AR, Boulouis A, Ludwig M, Lunn JE, Borghi GL, Schlereth A, Guenther M, Stitt M. 2019. Metabolite profiles reveal inter-specific variation in operation of the Calvin–Benson cycle in both C4 and C3 plants. Journal of Experimental Botany 70, 1843–1858.SNS

Chloroplast development in green plant tissues: the interplay between light, hormone, and transcriptional regulation.

Chloroplasts are best known for their role in photosynthesis, but they also allow nitrogen and sulphur assimilation, amino acid, fatty acid, nucleotide and hormone synthesis. How chloroplasts develop is therefore relevant to these diverse and fundamental biological processes, but also to attempts at their rational redesign. Light is strictly required for chloroplast formation in all angiosperms and directly regulates the expression of hundreds of chloroplast-related genes. Light also modulates the levels of several hormones including brassinosteriods, cytokinins, auxins and gibberellins, which themselves control chloroplast development particularly during early stages of plant development. Transcription factors such as GOLDENLIKE1&2 (GLK1&2), GATA NITRATE-INDUCIBLE CARBON METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA FACTOR 1 (CGA1) act downstream of both light and phytohormone signalling to regulate chloroplast development. Thus, in green tissues transcription factors, light signalling and hormone signalling form a complex network regulating the transcription of chloroplast- and photosynthesis-related genes to control the development and number of chloroplasts per cell. We use this conceptual framework to identify points of regulation that could be harnessed to modulate chloroplast abundance and increase photosynthetic efficiency of crops, and to highlight future avenues to overcome gaps in current knowledge

Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis.

Publication status: PublishedIn leaves of C4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C4 grasses, proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known whether C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How M and BS cells in C4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives. The M-BS interface of C4 G. gynandra showed higher plasmodesmal frequency compared with closely related C3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. We conclude that enhanced formation of plasmodesmata between M and BS cells is wired to the induction of photosynthesis in C4 G. gynandra

Plasmodesmal connectivity in C₄ Gynandropsis gynandra is induced by light and dependent on photosynthesis

In leaves of C₄ plants the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C₄ acids in mesophyll cells and subsequent decarboxylation in the bundle sheath. In C₄ grasses proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known if C₄ dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C₄ photosynthesis is not clear. How mesophyll and bundle sheath cells in C₄ leaves become highly connected is also not known. We investigated these questions using 3D- and 2D- electron microscopy on the C₄ dicotyledon Gynandropsis gynandra as well as phylogenetically close C₃ relatives. The mesophyll-bundle sheath interface of C₄ G. gynandra showed higher plasmodesmal frequency compared with closely related C₃ species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed chloroplast development or photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. We conclude that enhanced formation of plasmodesmata between mesophyll and bundle sheath cells is wired to the induction of photosynthesis in C₄ G. gynandra

C4 gene induction during de-etiolation evolved through changes in cis to allow integration with ancestral C3 gene regulatory networks.

C4 photosynthesis has evolved by repurposing enzymes found in C3 plants. Compared with the ancestral C3 state, accumulation of C4 cycle proteins is enhanced. We used de-etiolation of C4 Gynandropsis gynandra and C3 Arabidopsis thaliana to understand this process. C4 gene expression and chloroplast biogenesis in G. gynandra were tightly coordinated. Although C3 and C4 photosynthesis genes showed similar induction patterns, in G. gynandra, C4 genes were more strongly induced than orthologs from A. thaliana. In vivo binding of TGA and homeodomain as well as light-responsive elements such as G- and I-box motifs were associated with the rapid increase in transcripts of C4 genes. Deletion analysis confirmed that regions containing G- and I-boxes were necessary for high expression. The data support a model in which accumulation of transcripts derived from C4 photosynthesis genes in C4 leaves is enhanced because modifications in cis allowed integration into ancestral transcriptional networks

Shedding light on AT1G29480 of Arabidopsis thaliana-An enigmatic locus restricted to Brassicacean genomes.

A key feature of C4 Kranz anatomy is the presence of an enlarged, photosynthetically highly active bundle sheath whose cells contain large numbers of chloroplasts. With the aim to identify novel candidate regulators of C4 bundle sheath development, we performed an activation tagging screen with Arabidopsis thaliana. The reporter gene used encoded a chloroplast-targeted GFP protein preferentially expressed in the bundle sheath, and the promoter of the C4 phosphoenolpyruvate carboxylase gene from Flaveria trinervia served as activation tag because of its activity in all chlorenchymatous tissues of A. thaliana. Primary mutants were selected based on their GFP signal intensity, and one stable mutant named kb-1 with a significant increase in GFP fluorescence intensity was obtained. Despite the increased GFP signal, kb-1 showed no alterations to bundle sheath anatomy. The causal locus, AT1G29480, is specific to the Brassicaceae with its second exon being conserved. Overexpression and reconstitution studies confirmed that AT1G29480, and specifically its second exon, were sufficient for the enhanced GFP phenotype, which was not dependent on translation of the locus or its parts into protein. We conclude, therefore, that the AT1G29480 locus enhances the GFP reporter gene activity via an RNA-based mechanism

Shedding light on AT1G29480 of <scp> <i>Arabidopsis thaliana</i> </scp> —An enigmatic locus restricted to Brassicacean genomes

Abstract: A key feature of C4 Kranz anatomy is the presence of an enlarged, photosynthetically highly active bundle sheath whose cells contain large numbers of chloroplasts. With the aim to identify novel candidate regulators of C4 bundle sheath development, we performed an activation tagging screen with Arabidopsis thaliana . The reporter gene used encoded a chloroplast‐targeted GFP protein preferentially expressed in the bundle sheath, and the promoter of the C4 phosphoenolpyruvate carboxylase gene from Flaveria trinervia served as activation tag because of its activity in all chlorenchymatous tissues of A. thaliana . Primary mutants were selected based on their GFP signal intensity, and one stable mutant named kb‐1 with a significant increase in GFP fluorescence intensity was obtained. Despite the increased GFP signal, kb‐1 showed no alterations to bundle sheath anatomy. The causal locus, AT1G29480, is specific to the Brassicaceae with its second exon being conserved. Overexpression and reconstitution studies confirmed that AT1G29480, and specifically its second exon, were sufficient for the enhanced GFP phenotype, which was not dependent on translation of the locus or its parts into protein. We conclude, therefore, that the AT1G29480 locus enhances the GFP reporter gene activity via an RNA‐based mechanism