Maize is a globally important staple crop, providing food, animal feed, and industrial raw materials. Its extensive native diversity can be utilized to broaden the genetic basis for quantitative trait improvement and advance our understanding of the genotype-phenotype relationships underlying complex trait variation. In a forward genetic approach, we dissect a locus on chromosome 10 with major effects on Photosystem II (PSII) maximum quantum efficiency (Fv/Fm), non-photochemical quenching (NPQ) and biomass accumulation during early growth stages. Integrating molecular and physiological information we show that allelic variation at the gene encoding LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN6 (LHCB6 also known as CP24), a component of the PSII light-harvesting complex (LHCII) antenna, underlies this locus. We demonstrate that the allelic variation results from a hAT transposon insertion at lhcb6 and is associated with decreased accumulation of the LHCII antenna components LHCB6 and LHCB3. Based on proteomic analyses we propose candidate genes that partially compensate for the unfavorable early growth effects caused by impaired LHCII antenna assembly. Our work provides novel insights into the function of lhcb6 in the C4 crop maize and demonstrates the value of natural variation for the understanding and genetic improvement of complex photosynthetic processes.

The sustainability of maize cultivation would benefit tremendously from early sowing, but is hampered by low temperatures during early development in temperate climates. We show that allelic variation within the gene encoding subunit M of the NADH-dehydrogenase-like (NDH) complex (ndhm1) in a European maize landrace affects several quantitative traits that are relevant during early development in cold climates through NDH-mediated cyclic electron transport around photosystem I, a process crucial for photosynthesis and photoprotection. Beginning with a genome-wide association study for maximum potential quantum yield of photosystem II in dark-adapted leaves (Fv/Fm), we capitalized on the large phenotypic effects of a hAT transposon insertion in ndhm1 on multiple quantitative traits (early plant height [EPH], Fv/Fm, chlorophyll content, and cold tolerance) caused by the reduced protein levels of NDHM and associated NDH components. Analysis of the ndhm1 native allelic series revealed a rare allele of ndhm1 that is associated with small albeit significant improvements of Fv/Fm, photosystem II efficiency in light-adapted leaves (Phi PSII), and EPH compared with common alleles. Our work showcases the extraction of favorable alleles from locally adapted landraces, offering an efficient strategy for broadening the genetic variation of elite germplasm by breeding or genome editing. A European maize landrace harbors favorable allelic diversity in a gene encoding a subunit of the NADH-dehydrogenase-like complex, which can be harnessed to improve quantitative traits in maize.

Targeted utilization of native genetic diversity can expand the genetic basis of traits that exhibit limited genetic variation in elite breeding material and can enhance our understanding of the genotype-phenotype relationships associated with complex traits in crops. In a genome-wide association study in a European maize landrace we identified quantitative trait loci (QTL) that affected the maximum quantum efficiency of photosystem II (Fv/Fm) in field experiments. In a forward genetic approach, we focused on a QTL on chromosome 10, explaining a large proportion of the genetic variance for Fv/Fm in growth stages V4 (35%) and V6 (47%), for genetic dissection and candidate gene discovery. Integrating molecular and physiological information we show that allelic variation at the gene encoding LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN6 (LHCB6), a component of the photosystem II (PSII) light-harvesting complex (LHCII) antenna, underlies the variation in Fv/Fm. We demonstrate that the allelic variation results from a hAT transposon insertion at lhcb6 and is associated with differential accumulation of the LHCII antenna components LHCB6 and LHCB3, leading to differences in non-photochemical quenching (NPQ) and plant biomass accumulation. Based on proteomic analyses we propose candidate genes that compensate the unfavorable effects caused by impaired LHCII antenna assembly. Our work provides novel insights into the function of lhcb6 in the context of LHCII antenna assembly and demonstrates the value of natural variation in landraces for the understanding and genetic improvement of complex photosynthetic processes. ### Competing Interest Statement The authors have declared no competing interest. Federal Ministry of Education and Research, 031B0195, 031B0882, 031B1301

C4 species have evolved more than 60 times independently from C3 ancestors. This multiple and parallel evolution of the complex C4 trait indicates common underlying evolutionary mechanisms that might be identified by comparative analysis of closely related C3 and C4 species. Efficient C4 function depends on a distinctive leaf anatomy that is characterized by enlarged, chloroplast rich bundle sheath cells and a narrow vein spacing. To elucidate molecular mechanisms generating this so called Kranz anatomy, we analyzed a developmental series of leaves from the C4 plant Flaveria bidentis and the closely related C3 species Flaveria robusta using leaf clearing and whole transcriptome sequencing. Applying non-negative matrix factorization on the data identified four different zones with distinct transcriptome patterns in growing leaves of both species. Comparing these transcriptome patterns revealed an important role of auxin metabolism and especially auxin homeostasis for establishing the high vein density typical for C4 leaves.

SUMMARYC4 species have evolved more than 60 times independently from C3 ancestors. This multiple and parallel evolution of the complex C4 trait suggests common underlying evolutionary mechanisms, which could be identified by comparative analysis of closely related C3 and C4 species. Efficient C4 function depends on a distinctive leaf anatomy that is characterised by enlarged, chloroplast‐rich bundle sheath cells and narrow vein spacing. To elucidate the molecular mechanisms that generate the Kranz anatomy, we analysed a developmental series of leaves from the C4 plant Flaveria bidentis and the closely related C3 species Flaveria robusta by comparing anatomies and transcriptomes. Vascular density measurements of all nine leaf developmental stages identified three leaf anatomical zones whose proportions vary with respect to the developmental stage. We then deconvoluted the transcriptome datasets using non‐negative matrix factorisation, which identified four distinct transcriptome patterns in the growing leaves of both species. By integrating the leaf anatomy and transcriptome data, we were able to correlate the different transcriptional profiles with different developmental zones in the leaves. These comparisons revealed an important role for auxin metabolism, in particular auxin homeostasis (conjugation and deconjugation), in establishing the high vein density typical of C4 species.

Because of their photosynthetic capacity, leaves function as solar panels providing the basis for the growth of the entire plant. Although the molecular mechanisms of leaf development have been well studied in model dicot and monocot species, a lot of information is still needed about the interplay of the genes that regulate cell division and differentiation and thereby affect the photosynthetic performance of the leaf. We were specifically interested in understanding the differentiation of mesophyll and bundle sheath cells in Arabidopsis thaliana and aimed to identify genes that are involved in determining bundle sheath anatomy. To this end, we established a forward genetic screen by using ethyl methanesulfonate (EMS) for mutagenizing a reporter line expressing a chloroplast-targeted green fluorescent protein (sGFP) under the control of a bundle sheath-specific promoter. Based on the GFP fluorescence phenotype, numerous mutants were produced, and by pursuing a mapping-by-sequencing approach, the genomic segments containing mutated candidate genes were identified. One of the lines with an enhanced GFP fluorescence phenotype (named ELEVATED BUNDLE SHEATH CELLS SIGNAL 1 [ebss1]) was selected for further study, and the responsible gene was verified by CRISPR/Cas9-based mutagenesis of candidate genes located in the mapped genomic segment. The verified gene, At2g25970, encodes a K homology (KH) domain-containing protein.

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.

The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant-derived products. In the coming years, plant-based research will be among the major drivers ensuring food security and the expansion of the bio-based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity.

C4 photosynthesis is a remarkable complex trait, elucidations of the evolutionary trajectory of C4 photosynthesis from its ancestral C3 pathway can help us better understand the generic principles of the evolution of complex traits and guide the engineering of C3 crops for higher yields. Here, we used the genus Flaveria that contains C3, C3–C4, C4-like and C4 species as a system to study the evolution of C4 photosynthesis. We first mapped transcript abundance, protein sequence and morphological features onto the phylogenetic tree of the genus Flaveria, and calculated the evolutionary correlation of different features; we then predicted the relative changes of ancestral nodes of those features to illustrate the major events during the evolution of C4 photosynthesis. We found that gene expression and protein sequence showed consistent modification patterns in the phylogenetic tree. High correlation coefficients ranging from 0.46 to 0.9 among gene expression, protein sequence and morphology were observed. The greatest modification of those different features consistently occurred at the transition between C3-C4 species and C4-like species. Our results show highly coordinated changes in gene expression, protein sequence and morphological features, which support evolutionary major events during the evolution of C4 metabolism.

C-4 plants are believed to have evolved from C-3 plants through various C-3-C-4 intermediate stages in which a photorespiration-dependent CO2 concentration system known as C-2 photosynthesis operates. Genes involved in the C-4 cycle were thought to be recruited from orthologs present in C-3 species and developed cell-specific expression during C4 evolution. To understand the process of establishing C-4 photosynthesis, we performed whole-genome sequencing and investigated expression and mesophyll- or bundle-sheath-cell-specific localization of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), pyruvate, orthophosphate dikinase (PPDK) in C-3, C-3-C-4 intermediate, C-4-like, and C-4 Flaveria species. While genome sizes vary greatly, the number of predicted protein-coding genes was similar among C-3, C-3-C-4 intermediate, C4-like, and C4 Flaveria species. Cell-specific localization of the PEPC, NADP-ME, and PPDK transcripts was insignificant or weak in C-3-C-4 intermediate species, whereas these transcripts were expressed celltype specific in C-4-like species. These results showed that elevation of gene expression and cell-specific control of pre-existing C-4 cycle genes in C-3 species was involved in C-4 evolution. Gene expression was gradually enhanced during C-4 evolution, whereas cell-specific control was gained independently of quantitative transcriptional activation during evolution from C-3-C-4 intermediate to C-4 photosynthesis in genus Flaveria.

While the demand for paper and packaging material is increasing, industry and consumers are searching for more sustainable raw materials. This study evaluates three non-wood perennials to find alternative raw materials for the paper and pulp industry. Meadow hay, cup plant (Silphium perfoliatum L.) and Virginia mallow (Sida hermaphrodita (L.) Rusby) are attracting attention as potential raw materials for the bioeconomy due to biodiversity benefits, low fertilizer requirements as well as high yields. For the first time a detailed view on their fibre morphology and use as paper feedstock is given. After three different mechanical grinding methods, the plant material was screened, pulped in NaOH and beaten in a PFI mill. Birch fibre has been chosen as short-fibre control and blend base. Hand-sheets with different pulp blends of birch and one of the three raw materials were made, and paper properties were measured. For meadow hay, Virginia mallow and cup plant fibre lengths of 0.5, 1.3, and 0.9 mm were measured. Therefore, all perennial plant materials have comparable fibre lengths to hardwoods. Meadow hay blends with birch pulp percentages of 50 % and 75 % generated higher paper strength compared to the pure birch paper at a beating intensity of 5000 revolutions (PFI). The paper strength of cup plant and Virginia mallow blends is comparable to the strength of the birch control. Due to these promising results, all analyzed raw materials could find their application, especially in the growing area of sustainable packaging materials.

Udo Gowik: Institute of Plant Molecular and Developmental Biology, Heinrich-Heine-University, Dusseldorf, Germany, gowik@uni-duesseldorf.de. Steve Kelly: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom, steven.kelly@plants.ox.ac.uk. Sarah Covshoff: Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom, sarahcovshoff@gmail.com. Harmony Clayton: School of Molecular Sciences, University of Western Australia, Crawley, WA, Australia, 20152857@student.uwa.edu.au. Julian M. Hibberd: Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom, jmh65@cam.ac.uk. Rowan F. Sage: Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Canada, r.sage@utoronto.ca. Martha Ludwig: School of Molecular Sciences, University of Western Australia, Crawley, WA, Australia, martha.ludwig@uwa.edu.au. Gane Ka-Shu Wong: BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China; Department of Biological Sciences, University of Alberta, Edmonton AB, T6G 2E9, Canada; Department of Medicine, University of Alberta, Edmonton AB, T6G 2E1, Canada, gane@ualberta.ca. Peter Westhoff: Institute of Plant Molecular and Developmental Biology, Heinrich-Heine-University, Dusseldorf, Germany, west@uni-duesseldorf.de.

In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.

Cup plant (Silphium perfoliatum L.) represents a promising alternative to silage maize as an energy crop for biogas production. This non-food plant possesses a highly ecological value due to its long blooming period, ability to grow in low-input agriculture and positive influence on soil structure. So far, there have been almost no breeding attempts for the cup plant, and all field experiments showing its high biomass yield were conducted by using only a few cultivated populations of unclear ancestry. Comprehensive assessment of five such populations for their biomass and methane yield parameters revealed substantial genetic variations indicating the possibility of improving these traits through selection and breeding. Higher biomass yield is likely to be achieved by breeding for secondary traits such as plant height, shoot diameter and internode number as well as photoperiod response. For increasing the methane production, reduced lignin or fibre content in the biomass seems to be important. Genetic relationships among the populations were estimated using tunable genotyping by sequencing (tGBS) technology. Genetic structure and phylogeny analyses revealed that all the plants belong to the same gene pool and share a common ancestry. Four out of five populations demonstrate a low genetic differentiation, whereas the fifth one represents a clear example of population stratification. To achieve a successful domestication and breeding of this new high-yielding perennial crop, a broader base of genetic diversity needs to be ensured and complemented by innovative breeding strategies driven by molecular genetic and modern genomics approaches.

C-4 photosynthetic plants have evolved from C-3 ancestors and are characterized by differential expression of several hundred genes. Strict compartmentalization of key C-4 enzymes either to mesophyll (M) or bundle sheath cells is considered a crucial step towards the evolution of C-4 photosynthesis. In this study, we demonstrate that the 5 '-flanking sequences of the C-4 type phosphoenolpyruvate carboxylase (Ppc) gene from three C-4 grass species could drive M-cell-specific expression of a reporter gene in rice. In addition to that, we identified about 450 bp (upstream of their transcription start site) of the analyzed C(4)Ppc promoters contain all the essential regulatory elements for driving M-cell-specific expression in rice leaves. Importantly, four motifs of conserved nucleotide sequences (CNSs) were also determined, which are essential for the activity of the promoter. A putative interaction between the CNSs and an unknown upstream element(s) is required for driving M-cell-specific expression. This work identifies the evolutionary conservation of C(4)Ppc regulatory mechanisms of multiple closely related C-4 grass species.