Targeted A-to-G base editing in the organellar genomes of Arabidopsis with monomeric programmable deaminases.

Plastids and mitochondria are 2 intracellular organelles containing DNA-encoding partial but essential components for their roles, photosynthesis, and respiration. Precise base editing in both plastid and mitochondrial genomes would benefit their gene functional analysis and crop breeding. Targeted base editing in organellar genomes relies on a protein-based genome-editing system that uses the TALE-DNA recognition motif with deaminases. This is because the efficient delivery of guide RNA for clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems into organelles is currently impossible. Since TALE-based base editors used in organellar genomes are usually dimeric types, in this study, we used targeted A-to-G base editing in Arabidopsis (Arabidopsis thaliana) plastid and mitochondrial genomes with monomeric TALE-based deaminase for easier assembling of vectors. As a result, inheritable targeted A-to-G base editing of adenosine triphosphatase subunit 6-2 (atp6-2) in plant mitochondrial genomes and of 16S ribosomal RNA (16S rRNA) in plastid genomes of Arabidopsis was successfully induced by monomeric TALE-based adenine deaminase (AD) without off-target mutations. The monomeric TALE-based adenine deaminases also demonstrated a preference for editing the 8th T on the same strand from the recognition end. Phenotypic analysis showed that A-to-G conversion at 1139A of plastid 16S rRNA conferred substantial spectinomycin resistance in Arabidopsis, but not the other 2 potential-resistant mutations at 1131T and 1137T, predicted from the previous bacterial data. Our study demonstrated the feasibility of monomeric TALE-based ADs in plant organelles and their potential contribution to the functional analyses of plant organelles with easier assembling.

Characterization and development of a plastid genome base editor, ptpTALECD.

The modification of photosynthesis-related genes in plastid genomes may improve crop yields. Recently, we reported that a plastid-targeting base editor named ptpTALECD, in which a cytidine deaminase DddA functions as the catalytic domain, can homoplasmically substitute a targeted C to T in plastid genomes of Arabidopsis thaliana. However, some target Cs were not substituted. In addition, although ptpTALECD could substitute Cs on the 3' side of T and A, it was unclear whether it could also substitute Cs on the 3' side of G and C. In this study, we identified the preferential positions of the substituted Cs in ptpTALECD-targeting sequences in the Arabidopsis plastid genome. We also found that ptpTALECD could substitute Cs on the 3' side of all four bases in plastid genomes of Arabidopsis. More recently, a base editor containing an improved version of DddA (DddA11) was reported to substitute Cs more efficiently, and to substitute Cs on the 3' side of more varieties of bases in human mitochondrial genomes than a base editor containing DddA. Here, we also show that ptpTALECD_v2, in which a modified version of DddA11 functions as the catalytic domain, more frequently substituted Cs than ptpTALECD in the Arabidopsis plastid genome. We also found that ptpTALECD_v2 tended to substitute Cs at more positions than ptpTALECD. Our results reveal that ptpTALECD can cause a greater variety of codon changes and amino acid substitutions than previously thought, and that ptpTALECD and ptpTALECD_v2 are useful tools for the targeted base editing of plastid genomes.

Mitochondrial gene defects in Arabidopsis can broadly affect mitochondrial gene expression through copy number.

How mitochondria regulate the expression of their genes is poorly understood, partly because methods have not been developed for stably transforming mitochondrial genomes. In recent years, the disruption of mitochondrial genes has been achieved in several plant species using mitochondria-localized TALEN (mitoTALEN). In this study, we attempted to disrupt the NADH dehydrogenase subunit7 (NAD7) gene, a subunit of respiratory chain complex I, in Arabidopsis (Arabidopsis thaliana) using the mitoTALEN method. In some of the transformants, disruption of NAD7 was accompanied by severe growth inhibition and lethality, suggesting that NAD7 has an essential function in Arabidopsis. In addition, the mitochondrial genome copy number and overall expression of genes encoding mitochondrial proteins were generally increased by nad7 knockout. Similar increases were also observed in mutants with decreased NAD7 transcripts and with dysfunctions of other mitochondrial respiratory complexes. In these mutants, the expression of nuclear genes involved in mitochondrial translation or protein transport was induced in sync with mitochondrial genes. Mitochondrial genome copy number was also partly regulated by the nuclear stress-responsive factors NAC domain containing protein 17 and Radical cell death 1. These findings suggest the existence of overall gene-expression control through mitochondrial genome copy number in Arabidopsis and that disruption of single mitochondrial genes can have additional broad consequences in both the nuclear and mitochondrial genomes.

Targeted base editing in the mitochondrial genome of Arabidopsis thaliana.

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.

DOMINANT AWN INHIBITOR Encodes the ALOG Protein Originating from Gene Duplication and Inhibits AWN Elongation by Suppressing Cell Proliferation and Elongation in Sorghum.

The awn, a needle-like structure extending from the tip of the lemma in grass species, plays a role in environmental adaptation and fitness. In some crops, awns appear to have been eliminated during domestication. Although numerous genes involved in awn development have been identified, several dominant genes that eliminate awns are also known to exist. For example, in sorghum (Sorghum bicolor), the dominant awn-inhibiting gene has been known since 1921; however, its molecular features remain uncharacterized. In this study, we conducted quantitative trait locus analysis and a genome-wide association study of awn-related traits in sorghum and identified DOMINANT AWN INHIBITOR (DAI), which encodes the ALOG family protein on chromosome 3. DAI appeared to be present in most awnless sorghum cultivars, likely because of its effectiveness. Detailed analysis of the ALOG protein family in cereals revealed that DAI originated from a duplication of its twin paralog (DAIori) on chromosome 10. Observations of immature awns in near-isogenic lines revealed that DAI inhibits awn elongation by suppressing both cell proliferation and elongation. We also found that only DAI gained a novel function to inhibit awn elongation through an awn-specific expression pattern distinct from that of DAIori. Interestingly, heterologous expression of DAI with its own promoter in rice inhibited awn elongation in the awned cultivar Kasalath. We found that DAI originated from gene duplication, providing an interesting example of gain-of-function that occurs only in sorghum but shares its functionality with rice and sorghum.

Sorghum Ionomics Reveals the Functional SbHMA3a Allele that Limits Excess Cadmium Accumulation in Grains.

Understanding uptake and redistribution of essential minerals or sequestering of toxic elements is important for optimized crop production. Although the mechanisms controlling mineral transport have been elucidated in rice and other species, little is understood in sorghum-an important C4 cereal crop. Here, we assessed the genetic factors that govern grain ionome profiles in sorghum using recombinant inbred lines (RILs) derived from a cross between BTx623 and NOG (Takakibi). Pairwise correlation and clustering analysis of 22 elements, measured in sorghum grains harvested under greenhouse conditions, indicated that the parental lines, as well as the RILs, show different ionomes. In particular, BTx623 accumulated significantly higher levels of cadmium (Cd) than NOG, because of differential root-to-shoot translocation factors between the two lines. Quantitative trait locus (QTL) analysis revealed a prominent QTL for grain Cd concentration on chromosome 2. Detailed analysis identified SbHMA3a, encoding a P1B-type ATPase heavy metal transporter, as responsible for low Cd accumulation in grains; the NOG allele encoded a functional HMA3 transporter (SbHMA3a-NOG) whose Cd-transporting activity was confirmed by heterologous expression in yeast. BTx623 possessed a truncated, loss-of-function SbHMA3a allele. The functionality of SbHMA3a in NOG was confirmed by Cd concentrations of F2 grains derived from the reciprocal cross, in which the NOG allele behaved in a dominant manner. We concluded that SbHMA3a-NOG is a Cd transporter that sequesters excess Cd in root tissues, as shown in other HMA3s. Our findings will facilitate the isolation of breeding cultivars with low Cd in grains or in exploiting high-Cd cultivars for phytoremediation.

Spatial kernel models capturing field heterogeneity for accurate estimation of genetic potential.

According to Fisher's principles, an experimental field is typically divided into multiple blocks for local control. Although homogeneity is supposed within a block, this assumption may not be practical for large blocks, such as those including hundreds of plots. In line evaluation trials, which are essential in plant breeding, field heterogeneity must be carefully treated, because it can cause bias in the estimation of genetic potential. To more accurately estimate genotypic values in a large field trial, we developed spatial kernel models incorporating genome-wide markers, which consider continuous heterogeneity within a block and over the field. In the simulation study, the spatial kernel models were robust under various conditions. Although heritability, spatial autocorrelation range, replication number, and missing plots directly affected the estimation accuracy of genotypic values, the spatial kernel models always showed superior performance over the classical block model. We also employed these spatial kernel models for quantitative trait locus mapping. Finally, using field experimental data of bioenergy sorghum lines, we validated the performance of the spatial kernel models. The results suggested that a spatial kernel model is effective for evaluating the genetic potential of lines in a heterogeneous field.

NB-LRR-encoding genes conferring susceptibility to organophosphate pesticides in sorghum.

Organophosphate is the commonly used pesticide to control pest outbreak, such as those by aphids in many crops. Despite its wide use, however, necrotic lesion and/or cell death following the application of organophosphate pesticides has been reported to occur in several species. To understand this phenomenon, called organophosphate pesticide sensitivity (OPS) in sorghum, we conducted QTL analysis in a recombinant inbred line derived from the Japanese cultivar NOG, which exhibits OPS. Mapping OPS in this population identified a prominent QTL on chromosome 5, which corresponded to Organophosphate-Sensitive Reaction (OSR) reported previously in other mapping populations. The OSR locus included a cluster of three genes potentially encoding nucleotide-binding leucine-rich repeat (NB-LRR, NLR) proteins, among which NLR-C was considered to be responsible for OPS in a dominant fashion. NLR-C was functional in NOG, whereas the other resistant parent, BTx623, had a null mutation caused by the deletion of promoter sequences. Our finding of OSR as a dominant trait is important not only in understanding the diversified role of NB-LRR proteins in cereals but also in securing sorghum breeding free from OPS.

Targeted base editing in the plastid genome of Arabidopsis thaliana.

Bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases was recently reported to transiently substitute a targeted C to a T in mitochondrial DNA of mammalian cultured cells1. We applied this system to targeted base editing in the Arabidopsis thaliana plastid genome. The targeted Cs were homoplasmically substituted to Ts in some plantlets of the T1 generation and the mutations were inherited by their offspring independently of their nuclear-introduced vectors.

Genetic dissection of QTLs associated with spikelet-related traits and grain size in sorghum.

Although spikelet-related traits such as size of anther, spikelet, style, and stigma are associated with sexual reproduction in grasses, no QTLs have been reported in sorghum. Additionally, there are only a few reports on sorghum QTLs related to grain size, such as grain length, width, and thickness. In this study, we performed QTL analyses of nine spikelet-related traits (length of sessile spikelet, pedicellate spikelet, pedicel, anther, style, and stigma; width of sessile spikelet and stigma; and stigma pigmentation) and six grain-related traits (length, width, thickness, length/width ratio, length/thickness ratio, and width/thickness ratio) using sorghum recombinant inbred lines. We identified 36 and 7 QTLs for spikelet-related traits and grain-related traits, respectively, and found that most sorghum spikelet organ length- and width-related traits were partially controlled by the dwarf genes Dw1 and Dw3. Conversely, we found that these Dw genes were not strongly involved in the regulation of grain size. The QTLs identified in this study aid in understanding the genetic basis of spikelet- and grain-related traits in sorghum.

Climate-smart crops: key root anatomical traits that confer flooding tolerance.

Plants require water, but a deficit or excess of water can negatively impact their growth and functioning. Soil flooding, in which root-zone is filled with excess water, restricts oxygen diffusion into the soil. Global climate change is increasing the risk of crop yield loss caused by flooding, and the development of flooding tolerant crops is urgently needed. Root anatomical traits are essential for plants to adapt to drought and flooding, as they determine the balance between the rates of water and oxygen transport. The stele contains xylem and the cortex contains aerenchyma (gas spaces), which respectively contribute to water uptake from the soil and oxygen supply to the roots; this implies that there is a trade-off between the ratio of cortex and stele sizes with respect to adaptation to drought or flooding. In this review, we analyze recent advances in the understanding of root anatomical traits that confer drought and/or flooding tolerance to plants and illustrate the trade-off between cortex and stele sizes. Moreover, we introduce the progress that has been made in modelling and fully automated analyses of root anatomical traits and discuss how key root anatomical traits can be used to improve crop tolerance to soil flooding.

FMT, a protein that affects mitochondrial distribution, interacts with translation-related proteins in Arabidopsis thaliana.

KEY MESSAGE: Two translation-related proteins are identified as FMT-interacting proteins. However, FMT, unlike mutants of other CLU genes in fly and human, has no clear impact on the accumulation of mitochondrial proteins. Organelle distribution is critical for effective metabolism and stress response and is controlled by various environmental factors. Clustered mitochondria (CLU) superfamily genes affect mitochondrial distribution and their disruptions cause mitochondria to cluster within a cell in various species including yeast, fly, mammals and Arabidopsis. In Arabidopsis thaliana, Friendly mitochondria (FMT) is a CLU gene that is required for normal mitochondrial distribution, but its molecular function is unclear. Here, we demonstrate that FMT interacts with some translation-related proteins (translation initiation factor eIFiso4G1 and glutamyl-tRNA synthetase OVA9), as well as itself. We also show FMT forms dynamic particles in the cytosol that sometimes move with mitochondria, and their movements are mainly controlled by actin filaments but also by microtubules. Similar results have been reported for animal CLU orthologs. However, an fmt mutant, unlike animal clu mutants, did not show any clear decrease of nuclear-encoded mitochondrial protein levels. This difference may reflect a functional divergence of FMT from other CLU superfamily genes.

Key root traits of Poaceae for adaptation to soil water gradients.

Drought and flooding are contrasting abiotic stressors for plants. Evidence is accumulating for root anatomical traits being essential for the adaptation to drought or flooding. However, an integrated approach to comprehensively understand root anatomical traits has not yet been established. Here we analysed the root anatomical traits of 18 wild Poaceae species differing in adaptation to a range of soil water content. Regression model analyses revealed the optimal anatomical traits that were required by the plants to adapt to low or high soil water content. While the area and number of each root tissue (e.g. stele, cortex, xylem or aerenchyma) were not strongly correlated to the soil water content, the ratio of the root tissue areas (cortex to stele ratio (CSR), xylem to stele ratio (XSR) and aerenchyma to cortex ratio (ACR)) could fully explain the adaptations of the wild Poaceae species to the soil water gradients. Our results demonstrate that the optimal anatomical traits for the adaptations to soil water content can be determined by three indices (i.e. CSR, XSR and ACR), and thus we propose that these root anatomical indices can be used to improve the tolerance of crops to drought and flooding stresses.

Targeted gene disruption of ATP synthases 6-1 and 6-2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs.

We recently achieved targeted disruptions of cytoplasmic male sterility (CMS)-associated genes in the mitochondrial genomes of rice and rapeseed by using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). It was the first report of stable and heritable targeted gene modification of plant mitochondrial genomes. Here, we attempted to use mitoTALENs to disrupt two mitochondrial genes in the model plant Arabidopsis thaliana(Arabidopsis) using three different promoters and two types of TALENs. The targets were the two isoforms of the ATP synthase subunit 6 gene, atp6-1 and atp6-2. Each of these genes was successfully deleted and the mitochondrial genomes were recovered in a homoplasmic state. The nuclear genome also has a copy of atp6-1, and we were able to confirm that it was the mitochondrial gene and not the nuclear pseudogene that was knocked out. Among the three mitoTALEN promoters tried, the RPS5A promoter was the most effective. Conventional mitoTALENs were more effective than single-molecule mito-compactTALENs. Targeted mitochondrial gene deletion was achieved by crossing as well as by floral-dip transformation to introduce the mitoTALEN constructs into the nucleus. The gene disruptions were caused by large (kb-size) deletions. The ends of the remaining sequences were connected to distant loci, mostly by illegitimate homologous recombinations between repeats.

Dissecting the Genetic Architecture of Biofuel-Related Traits in a Sorghum Breeding Population.

In sorghum [Sorghum bicolor (L.) Moench], hybrid cultivars for the biofuel industry are desired. Along with selection based on testcross performance, evaluation of the breeding population per se is also important for the success of hybrid breeding. In addition to additive genetic effects, non-additive (i.e., dominance and epistatic) effects are expected to contribute to the performance of early generations. Unfortunately, studies on early generations in sorghum breeding programs are limited. In this study, we analyzed a breeding population for bioenergy sorghum, which was previously developed based on testcross performance, to compare genomic selection models both trained on and evaluated for the per se performance of the 3rd generation S0 individuals. Of over 200 ancestral inbred accessions in the base population, only 13 founders contributed to the 3rd generation as progenitors. Compared to the founders, the performances of the population per se were improved for target traits. The total genetic variance within the S0 generation progenies themselves for all traits was mainly additive, although non-additive variances contributed to each trait to some extent. For genomic selection, linear regression models explicitly considering all genetic components showed a higher predictive ability than other linear and non-linear models. Although the number and effect distribution of underlying loci was different among the traits, the influence of priors for marker effects was relatively small. These results indicate the importance of considering non-additive effects for dissecting the genetic architecture of early breeding generations and predicting the performance per se.

Longin R-SNARE is retrieved from the plasma membrane by ANTH domain-containing proteins in Arabidopsis.

The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.

Effect of salt tolerance on biomass production in a large population of sorghum accessions.

Salinity causes major reductions in cultivated land area, crop productivity, and crop quality, and salt-tolerant crops have been required to sustain agriculture in salinized areas. The annual C4 crop plant Sorghum bicolor (L.) Moench is salt tolerant, with large variation among accessions. Sorghum's salt tolerance is often evaluated during early growth, but such evaluations are weakly related to overall performance. Here, we evaluated salt tolerance of 415 sorghum accessions grown in saline soil (0, 50, 100, and 150 mM NaCl) for 3 months. Some accessions produced up to 400 g per plant of biomass and showed no growth inhibition at 50 mM NaCl. Our analysis indicated that the genetic factors that affected biomass production under 100 mM salt stress were more different from those without salt stress, comparing to the differences between those under 50 mM and 100 mM salt stress. A genome-wide association study for salt tolerance identified two single-nucleotide polymorphisms (SNPs) that were significantly associated with biomass production, only at 50 mM NaCl. Additionally, two SNPs were significantly associated with salt tolerance index as an indicator for growth response of each accession to salt stress. Our results offer candidate genetic resources and SNP markers for breeding salt-tolerant sorghum.

A Role for Auxin in Ethylene-Dependent Inducible Aerenchyma Formation in Rice Roots.

Internal oxygen diffusion from shoot to root tips is enhanced by the formation of aerenchyma (gas space) in waterlogged soils. Lysigenous aerenchyma is created by programmed cell death and subsequent lysis of the root cortical cells. Rice (Oryza sativa) forms aerenchyma constitutively under aerobic conditions and increases its formation under oxygen-deficient conditions. Recently, we have demonstrated that constitutive aerenchyma formation is regulated by auxin signaling mediated by Auxin/indole-3-acetic acid protein (AUX/IAA; IAA). While ethylene is involved in inducible aerenchyma formation, the relationship of auxin and ethylene during aerenchyma formation remains unclear. Here, we examined the effects of oxygen deficiency and ethylene on aerenchyma formation in the roots of a rice mutant (iaa13) in which auxin signaling is suppressed by a mutation in the degradation domain of IAA13 protein. The results showed that AUX/IAA-mediated auxin signaling contributes to ethylene-dependent inducible aerenchyma formation in rice roots. An auxin transport inhibitor abolished aerenchyma formation under oxygen-deficient conditions and reduced the expression of genes encoding ethylene biosynthesis enzymes, further supporting the idea that auxin is involved in ethylene-dependent inducible aerenchyma formation. Based on these studies, we propose a mechanism that underlies the relationship between auxin and ethylene during inducible aerenchyma formation in rice roots.

RAD-seq-Based High-Density Linkage Map Construction and QTL Mapping of Biomass-Related Traits in Sorghum using the Japanese Landrace Takakibi NOG.

Sorghum [Sorghum bicolor (L.) Moench] grown locally by Japanese farmers is generically termed Takakibi, although its genetic diversity compared with geographically distant varieties or even within Takakibi lines remains unclear. To explore the genomic diversity and genetic traits controlling biomass and other physiological traits in Takakibi, we focused on a landrace, NOG, in this study. Admixture analysis of 460 sorghum accessions revealed that NOG belonged to the subgroup that represented Asian sorghums, and it was only distantly related to American/African accessions including BTx623. In an attempt to dissect major traits related to biomass, we generated a recombinant inbred line (RIL) from a cross between BTx623 and NOG, and we constructed a high-density linkage map based on 3,710 single-nucleotide polymorphisms obtained by restriction-site-associated DNA sequencing of 213 RIL individuals. Consequently, 13 fine quantitative trait loci (QTLs) were detected on chromosomes 2, 3, 6, 7, 8 and 9, which included five QTLs for days to heading, three for plant height (PH) and total shoot fresh weight and two for Brix. Furthermore, we identified two dominant loci for PH as being identical to the previously reported dw1 and dw3. Together, these results corroborate the diversified genome of Japanese Takakibi, while the RIL population and high-density linkage map generated in this study will be useful for dissecting other important traits in sorghum.