Maize smart-canopy architecture enhances yield at high densities.

Increasing planting density is a key strategy for enhancing maize yields1-3. An ideotype for dense planting requires a 'smart canopy' with leaf angles at different canopy layers differentially optimized to maximize light interception and photosynthesis4-6, among other features. Here we identified leaf angle architecture of smart canopy 1 (lac1), a natural mutant with upright upper leaves, less erect middle leaves and relatively flat lower leaves. lac1 has improved photosynthetic capacity and attenuated responses to shade under dense planting. lac1 encodes a brassinosteroid C-22 hydroxylase that predominantly regulates upper leaf angle. Phytochrome A photoreceptors accumulate in shade and interact with the transcription factor RAVL1 to promote its degradation via the 26S proteasome, thereby inhibiting activation of lac1 by RAVL1 and decreasing brassinosteroid levels. This ultimately decreases upper leaf angle in dense fields. Large-scale field trials demonstrate that lac1 boosts maize yields under high planting densities. To quickly introduce lac1 into breeding germplasm, we transformed a haploid inducer and recovered homozygous lac1 edits from 20 diverse inbred lines. The tested doubled haploids uniformly acquired smart-canopy-like plant architecture. We provide an important target and an accelerated strategy for developing high-density-tolerant cultivars, with lac1 serving as a genetic chassis for further engineering of a smart canopy in maize.

Genetic regulation of self-organizing azimuthal canopy orientations and their impacts on light interception in maize.

The efficiency of solar radiation interception contributes to the photosynthetic efficiency of crop plants. Light interception is a function of canopy architecture, including plant density; leaf number, length, width, and angle; and azimuthal canopy orientation. We report on the ability of some maize (Zea mays) genotypes to alter the orientations of their leaves during development in coordination with adjacent plants. Although the upper canopies of these genotypes retain the typical alternate-distichous phyllotaxy of maize, their leaves grow parallel to those of adjacent plants. A genome-wide association study (GWAS) on this parallel canopy trait identified candidate genes, many of which are associated with shade avoidance syndrome, including phytochromeC2. GWAS conducted on the fraction of photosynthetically active radiation (PAR) intercepted by canopies also identified multiple candidate genes, including liguleless1 (lg1), previously defined by its role in ligule development. Under high plant densities, mutants of shade avoidance syndrome and liguleless genes (lg1, lg2, and Lg3) exhibit altered canopy patterns, viz, the numbers of interrow leaves are greatly reduced as compared to those of nonmutant controls, resulting in dramatically decreased PAR interception. In at least the case of lg2, this phenotype is not a consequence of abnormal ligule development. Instead, liguleless gene functions are required for normal light responses, including azimuth canopy re-orientation.

Participation of miR165a in the Phytochrome Signal Transduction in Maize (Zea mays L.) Leaves under Changing Light Conditions.

The involvement of the microRNA miR165a in the light-dependent mechanisms of regulation of target genes in maize (Zea mays) has been studied. The light-induced change in the content of free miR165a was associated with its binding by the AGO10 protein and not with a change in the rate of its synthesis from the precursor. The use of knockout Arabidopsis plants for the phytochrome A and B genes demonstrated that the presence of an active form of phytochrome B causes an increase in the level of the RNA-induced silencing miR165a complex, which triggers the degradation of target mRNAs. The two fractions of vesicles from maize leaves, P40 and P100 that bind miR165a, were isolated by ultracentrifugation. The P40 fraction consisted of larger vesicles of the size >0.170 µm, while the P100 fraction vesicles were <0.147 µm. Based on the quantitative PCR data, the predominant location of miR165a on the surface of extracellular vesicles of both fractions was established. The formation of the active form of phytochrome upon the irradiation of maize plants with red light led to a redistribution of miR165a, resulting in an increase in its proportion inside P40 vesicles and a decrease in P100 vesicles.

Rapid Birth or Death of Centromeres on Fragmented Chromosomes in Maize.

Comparative genomics has revealed common occurrences in karyotype evolution such as chromosomal end-to-end fusions and insertions of one chromosome into another near the centromere, as well as many cases of de novo centromeres that generate positional polymorphisms. However, how rearrangements such as dicentrics and acentrics persist without being destroyed or lost remains unclear. Here, we sought experimental evidence for the frequency and timeframe for inactivation and de novo formation of centromeres in maize (Zea mays). The pollen from plants with supernumerary B chromosomes was gamma-irradiated and then applied to normal maize silks of a line without B chromosomes. In ∼8,000 first-generation seedlings, we found many B-A translocations, centromere expansions, and ring chromosomes. We also found many dicentric chromosomes, but a fraction of these show only a single primary constriction, which suggests inactivation of one centromere. Chromosomal fragments were found without canonical centromere sequences, revealing de novo centromere formation over unique sequences; these were validated by immunolocalization with Thr133-phosphorylated histone H2A, a marker of active centromeres, and chromatin immunoprecipitation-sequencing with the CENH3 antibody. These results illustrate the regular occurrence of centromere birth and death after chromosomal rearrangement during a narrow window of one to potentially only a few cell cycles for the rearranged chromosomes to be recognized in this experimental regime.

Transcriptomic network analyses shed light on the regulation of cuticle development in maize leaves.

Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal that phyB1 phyB2 double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

Breeding effects on canopy light attenuation in maize: a retrospective and prospective analysis.

The light attenuation process within a plant canopy defines energy capture and vertical distribution of light and nitrogen (N). The vertical light distribution can be quantitatively described with the extinction coefficient (k), which associates the fraction of intercepted photosynthetically active radiation (fPARi) with the leaf area index (LAI). Lower values of k correspond to upright leaves and homogeneous vertical light distribution, increasing radiation use efficiency (RUE). Yield gains in maize (Zea mays L.) were accompanied by increases in optimum plant density and leaf erectness. Thus, the yield-driven breeding programs and management changes, such as reduced row spacing, selected a more erect leaf habit under different maize production systems (e.g., China and the USA). In this study, data from Argentina revealed that k decreased at a rate of 1.1% year-1 since 1989, regardless of plant density and in agreement with Chinese reports (1.0% year-1 since 1981). A reliable assessment of changes in k over time is critical for predicting (i) modifications in resource use efficiency (e.g. radiation, water, and N), improving estimations derived from crop simulation models; (ii) differences in productivity caused by management practices; and (iii) limitations to further exploit this trait with breeding.

Posttranslational Modification of the NADP-Malic Enzyme Involved in C4 Photosynthesis Modulates the Enzymatic Activity during the Day.

Evolution of the C4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C4 pathway. Here, we show that regulation of maize (Zea mays) C4-NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C4-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. Phosphorylation of ZmC4-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C4-NADP-ME indicated that Ser419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC4-NADP-ME at Ser419 during the first hours in the light is a cellular mechanism that fine tunes the enzymatic activity to coordinate the carbon concentration mechanism with the CO2 fixation rate, probably to avoid CO2 leakiness from bundle sheath cells.

Comparative transcriptome and metabolome analysis reveal glutathione metabolic network and functional genes underlying blue and red-light mediation in maize seedling leaf.

BACKGROUND: Light quality severely affects biosynthesis and metabolism-associated process of glutathione. However, the role of specific light is still unclear on the glutathione metabolism. In this article, comparatively transcriptome and metabolome methods are used to fully understand the blue and red-light conditions working on the glutathione metabolism in maize seedling leaf. RESULTS: There are 20 differently expressed genes and 4 differently expressed metabolites in KEGG pathway of glutathione metabolism. Among them, 12 genes belong to the glutathione S-transferase family, 3 genes belong to the ascorbate peroxidase gene family and 2 genes belong to the ribonucleoside-diphosphate reductase gene family. Three genes, G6PD, SPDS1, and GPX1 belong to the gene family of glucose 6-phosphate dehydrogenase, spermidine synthase, and glutathione peroxidase, respectively. Four differently expressed metabolites are identified. Three of them, Glutathione disulfide, Glutathione, and l-γ-Glutamyl-L-amino acid are decreased while L-Glutamate is increased. In addition, Through PPI analysis, two annotated genes gst16 and DAAT, and 3 unidentified genes 100381533, pco105094 and umc2770, identified as RPP13-like3, BCAT-like1and GMPS, were obtained. By the analysis of protein sequence and PPI network, we predict that pco105094 and umc2770 were involved in the GSSG-GSH and AsA-GSH cycle in the network of glutathione metabolism. CONCLUSIONS: Compared to red light, blue light remarkably changed the transcription signal transduction and metabolism of glutathione metabolism. Differently expressed genes and metabolic mapped to the glutathione metabolism signaling pathways. In total, we obtained three unidentified genes, and two of them were predicted in current glutathione metabolism network. This result will contribute to the research of glutathione metabolism of maize.

Subdivision of Light Signaling Networks Contributes to Partitioning of C4 Photosynthesis.

Plants coordinate the expression of photosynthesis-related genes in response to growth and environmental changes. In species that conduct two-cell C4 photosynthesis, expression of photosynthesis genes is partitioned such that leaf mesophyll and bundle sheath cells accumulate different components of the photosynthetic pathway. The identities of the regulatory networks that facilitate this partitioning are unknown. Here, we show that differences in light perception between mesophyll and bundle sheath cells facilitate differential regulation and accumulation of photosynthesis gene transcripts in the C4 crop maize (Zea mays). Key components of the photosynthesis gene regulatory network differentially accumulated between mesophyll and bundle sheath cells, indicative of differential network activity across cell types. We further show that blue (but not red) light is necessary and sufficient to activate photosystem II assembly in mesophyll cells in etiolated maize. Finally, we demonstrate that 61% of all light-induced mesophyll and bundle sheath genes were induced only by blue light or only by red light, but not both. These findings provide evidence that subdivision of light signaling networks is a component of cellular partitioning of C4 photosynthesis in maize.

Opposite response of maize ZmCCT to photoperiod due to transposon jumping.

KEY MESSAGE: The new 4.2-kb transposable insertion in the intron of ZmCCT reversely responded relative to the known 5.1-kb transposable insertion to photoperiods between low- and high-latitude regions. Flowering time is a key trait for cereal adaptation that is controlled by a complex genetic background in maize. The effect of multiple alleles from a quantitative trait locus (QTL) on flowering time remains largely unknown. Here, we fine-mapped a major QTL for flowering time on maize chromosome 10 corresponding to ZmCCT, where a new allele with a 4.2-kilobase (kb) transposable insertion was present in the intron. The known allele with a 5.1-kb transposon insertion in the promoter of ZmCCT enhances flowering in high-latitude regions, but has no effect on flowering time in low-latitude regions in comparison with the null allele lacking this insertion. However, our new allele with a 4.2-kb insertion reduced flowering in the low-latitude region, but produced unchanged flowering time in the high-latitude region relative to the 5.1-kb transposable insertion. Transcription analysis revealed that the new allele with 4.2-kb insertion versus the 5.1-kb insertion repressed and unchanged the transcription of ZmCCT in the low- and high-latitude regions, respectively. Thus, the allele with the 4.2-kb transposable insertion showed a completely opposite response to photoperiods between these two regions. Phylogenetic analysis revealed that the 4.2-kb transposable insertion in the two Northern flint corns originated from tropical maize. RNA-seq analysis and dual-luciferase transient expression assays further identified a conserved gene regulation network of ZmCCT between maize and rice, in which ZmCCT directly repressed the transcription of the florigen gene ZCN8 via ZmEhd1. Our results suggest that transposable elements play an important role in maize adaptation.

Dark response genes: a group of endogenous pendulum/timing players in maize?

MAIN CONCLUSION: Maize has a set of dark response genes, expression of which is influenced by multiple factor and varies with maize inbred lines but without germplasm specificity. The response to photoperiod is a common biological issue across the species kingdoms. Dark is as important as light in photoperiod. However, further in-depth understanding of responses of maize (Zea mays) to light and dark transition under photoperiod is hindered due to the lack of understanding of dark response genes. With multiple public "-omic" datasets of temperate and tropical/subtropical maize, 16 maize dark response genes, ZmDRGs, were found and had rhythmic expression under dark and light-dark cycle. ZmDRGs 6-8 were tandemly duplicated. ZmDRGs 2, 13, and 14 had a chromosomal collinearity with other maize genes. ZmDRGs 1-11 and 13-16 had copy-number variations. ZmDRGs 2, 9, and 16 showed 5'-end sequence deletion mutations. Some ZmDRGs had chromatin interactions and underwent DNA methylation and/or m6A mRNA methylation. Chromosomal histones associated with 15 ZmDRGs were methylated and acetylated. ZmDRGs 1, 2, 4, 9, and 13 involved photoperiodic phenotypes. ZmDRG16 was within flowering-related QTLs. ZmDRGs 1, 3, and 6-11 were present in cis-acting expression QTLs (eQTLs). ZmDRGs 1, 4, 6-9, 11, 12, and 14-16 showed co-expression with other maize genes. Some of ZmDRG-encoded ZmDRGs showed obvious differences in abundance and phosphorylation. CONCLUSION: Sixteen ZmDRGs 1-16 are associated with the dark response of maize. In the process of post-domestication and/or breeding, the ZmDRGs undergo the changes without germplasm specificity, including epigenetic modifications, gene copy numbers, chromatin interactions, and deletion mutations. In addition to effects by these factors, ZmDRG expression is influenced by promoter elements, cis-acting eQTLs, and co-expression networks.

Changes in the vertical distribution of leaf area enhanced light interception efficiency in maize over generations of selection.

Breeders select for yield, thereby indirectly selecting for traits that contribute to it. We tested if breeding has affected a range of traits involved in plant architecture and light interception, via the analysis of a panel of 60 maize hybrids released from 1950 to 2015. This was based on novel traits calculated from reconstructions derived from a phenotyping platform. The contribution of these traits to light interception was assessed in virtual field canopies composed of 3D plant reconstructions, with a model tested in a real field. Two categories of traits had different contributions to genetic progress. (a) The vertical distribution of leaf area had a high heritability and showed a marked trend over generations of selection. Leaf area tended to be located at lower positions in the canopy, thereby improving light penetration and distribution in the canopy. This potentially increased the carbon availability to ears, via the amount of light absorbed by the intermediate canopy layer. (b) Neither the horizontal distribution of leaves in the relation to plant rows nor the response of light interception to plant density showed appreciable trends with generations. Hence, among many architectural traits, the vertical distribution of leaf area was the main indirect target of selection.

Apigenin produced by maize flavone synthase I and II protects plants against UV-B-induced damage.

Flavones, one of the largest groups of flavonoids, have beneficial effects on human health and are considered of high nutritional value. Previously, we demonstrated that maize type I flavone synthase (ZmFNSI) is one of the enzymes responsible for the synthesis of O-glycosyl flavones in floral tissues. However, in related species such as rice and sorghum, type II FNS enzymes also contribute to flavone biosynthesis. In this work, we provide evidence that maize has both one FNSI and one FNSII flavone synthases. Arabidopsis transgenic plants expressing each FNS enzyme were generated to validate the role of flavones in protecting plants against UV-B radiation. Here, we demostrate that ZmCYP93G7 (FNSII) has flavone synthase activity and is able to complement the Arabidopsis dmr6 mutant, restoring the susceptibility to Pseudomonas syringae. ZmFNSII expression is controlled by the C1/PL1 + R/B anthocyanin transcriptional complexes, and both ZmFNSI and ZmFNSII are regulated by UV-B. Arabidopsis transgenic plants expressing ZmFNSI or ZmFNSII that accumulate apigenin exhibit less UV-B-induced damage than wild-type plants. Together, we show that maize has two FNS-type enzymes that participate in the synthesis of apigenin, conferring protection against UV-B radiation.

Response of plasmodesmata formation in leaves of C4 grasses to growth irradiance.

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C4 leaves is a key requirement for C4 photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C4 species using our PD quantification method, which combines three-dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP-ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 µmol m-2  s-1 ), and high light (1,000 µmol m-2  s-1 ), was observed both at seedling and established growth stages. We found that the effect of growth irradiance on C4 leaf PD density depended on plant age and species. The high light treatment resulted in two to four-fold greater PD density per unit leaf area than at low light, due to greater area of PD clusters and greater PD size in high light plants. These results along with our finding that the effect of light on M-BS PD density was not tightly linked to photosynthetic capacity suggest a complex mechanism underlying the dynamic response of C4 leaf PD formation to growth irradiance.

Increased photosensitivity at early growth as a possible mechanism of maize adaptation to cold springs.

Maize is a cold-sensitive species, but selective breeding programs have recently succeeded in producing plants strikingly well adapted to the cold springs of a temperate climate, showing the potential for improved cold tolerance. The aim of the present study was to determine whether the adaptation of some inbred lines to spring chills is due to their increased true cold tolerance or whether it only represents an avoidance mechanism, which was the sole mode of adaptation during early stages of agricultural dispersal of maize towards higher latitudes. By characterizing numerous physiological features of several lines of different cold sensitivity, we show that a combination of both avoidance and tolerance is involved. A novel avoidance mechanism was found that favored unhindered development of the photosynthetic apparatus through protection of the shoot apex below soil level due to a shortened mesocotyl. It seems to be mediated by increased seedling photosensitivity at early growth stages. True tolerance involved improved protection of the cell membrane against cold injury at temperatures close to 0 °C and stimulation of light-induced processes (accumulation of anthocyanins, carotenoids, and chlorophyll, proper development of chloroplasts) at temperatures in the range of 10-14 °C, likely also related to the increased photosensitivity and mediated by gibberellin signaling.

Relationship between fluorescence yield and photochemical yield under water stress and intermediate light conditions.

The dynamics between fluorescence (Fs) yield and photochemical (P) yield in a changing environment are essential for understanding the relationship between photosynthesis and fluorescence. The ratio of Fs yield and P yield tends to be constant under high light intensity, but the relationship between these two yields, and its response to environmental conditions, need to be explored further under intermediate and low light. In this study, we performed leaf-scale measurements of fluorescence parameters by pulse-amplitude modulation (PAM) technology in summer maize (Zea mays L.) plants grown under intermediate light conditions in a climate chamber. Plants were treated as moderately water stressed and non-water stressed. Results showed that a decrease in P yield was accompanied by increases in Fs yield and non-photochemical quenching (NPQ) yield in response to moderate water stress under intermediate and low light conditions. Fs yield was negatively correlated with P yield under intermediate and low light conditions when there was sufficient soil water in the root zone. Under water stress, the correlation between Fs yield and P yield was negative in low light, but became positive under higher light levels. Fs yield was negatively related to P yield when NPQ yield was low; however, they were synergistically and positively associated when excessive light dissipation was dominated by NPQ.

Low-fluence blue light-induced phosphorylation of Zmphot1 mediates the first positive phototropism.

Phototropin1 (phot1) perceives low- to high-fluence blue light stimuli and mediates both the first and second positive phototropisms. High-fluence blue light is known to induce autophosphorylation of phot1, leading to the second positive phototropism. However, the phosphorylation status of phot1 by low-fluence blue light that induces the first positive phototropism had not been observed. Here, we conducted a phosphoproteomic analysis of maize coleoptiles to investigate the fluence-dependent phosphorylation status of Zmphot1. High-fluence blue light induced phosphorylation of Zmphot1 at several sites. Notably, low-fluence blue light significantly increased the phosphorylation level of Ser291 in Zmphot1. Furthermore, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be more abundant in the low-fluence blue light-irradiated sides than in the shaded sides of coleoptiles. The roles of these phosphorylation events in phototropism were explored by heterologous expression of ZmPHOT1 in the Arabidopsis thaliana phot1phot2 mutant. The first positive phototropism was restored in wild-type ZmPHOT1-expressing plants; however, plants expressing S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 showed significantly reduced complementation rates. All transgenic plants tested in this study exhibited a normal second positive phototropism. These findings provide the first indication that low-fluence blue light induces phosphorylation of Zmphot1 and that this induced phosphorylation is crucial for the first positive phototropism.

The energy budget in C4 photosynthesis: insights from a cell-type-specific electron transport model.

Extra ATP required in C4 photosynthesis for the CO2 -concentrating mechanism probably comes from cyclic electron transport (CET). As metabolic ATP : NADPH requirements in mesophyll (M) and bundle-sheath (BS) cells differ among C4 subtypes, the subtypes may differ in the extent to which CET operates in these cells. We present an analytical model for cell-type-specific CET and linear electron transport. Modelled NADPH and ATP production were compared with requirements. For malic-enzyme (ME) subtypes, c. 50% of electron flux is CET, occurring predominantly in BS cells for standard NADP-ME species, but in a ratio of c. 6 : 4 in BS : M cells for NAD-ME species. Some C4 acids follow a secondary decarboxylation route, which is obligatory, in the form of 'aspartate-malate', for the NADP-ME subtype, but facultative, in the form of phosphoenolpyruvate-carboxykinase (PEP-CK), for the NAD-ME subtype. The percentage for secondary decarboxylation is c. 25% and that for 3-phosphoglycerate reduction in BS cells is c. 40%; but these values vary with species. The 'pure' PEP-CK type is unrealistic because its is impossible to fulfil ATP : NADPH requirements in BS cells. The standard PEP-CK subtype requires negligible CET, and thus has the highest intrinsic quantum yields and deserves further studies in the context of improving canopy productivity.

UV-B Inhibits Leaf Growth through Changes in Growth Regulating Factors and Gibberellin Levels.

Ultraviolet-B (UV-B) radiation affects leaf growth in a wide range of species. In this work, we demonstrate that UV-B levels present in solar radiation inhibit maize (Zea mays) leaf growth without causing any other visible stress symptoms, including the accumulation of DNA damage. We conducted kinematic analyses of cell division and expansion to understand the impact of UV-B radiation on these cellular processes. Our results demonstrate that the decrease in leaf growth in UV-B-irradiated leaves is a consequence of a reduction in cell production and a shortened growth zone (GZ). To determine the molecular pathways involved in UV-B inhibition of leaf growth, we performed RNA sequencing on isolated GZ tissues of control and UV-B-exposed plants. Our results show a link between the observed leaf growth inhibition and the expression of specific cell cycle and developmental genes, including growth-regulating factors (GRFs) and transcripts for proteins participating in different hormone pathways. Interestingly, the decrease in the GZ size correlates with a decrease in the concentration of GA19, the immediate precursor of the active gibberellin, GA1, by UV-B in this zone, which is regulated, at least in part, by the expression of GRF1 and possibly other transcription factors of the GRF family.