Search

Transposition of transposable elements affect expression levels, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus. For example, in grape, presence of the Gret1 retrotransposon in the promoter region of the VvMYBA1a allele at the VvMYBA1 locus suppress the expression of the VvMYBA1 transcription factor gene for the anthocyanin biosynthesis and this transposon insertion is responsible for the green berry skin color of Vitis labrascana, ‘Shine Muscat’, a major grape cultivar in Japan. To prove that transposons in grape genome can be removed by genome editing, we focused on Gret1 in the VvMYBA1a allele as a target of CRISPR/Cas9 mediated transposon removal. PCR amplification and sequencing detected Gret1 eliminated cells in 19 of 45 transgenic plants. Although we have not yet confirmed any effects on grape berry skin color, we were successful in demonstrating that cleaving the long terminal repeat (LTR) present at both ends of Gret1 can efficiently eliminate the transposon.

Abstract Background Sorghum (Sorghum bicolor L.) is used as a raw material for biofuels because it accumulates sugars at high levels in the stem. Lodging of sorghum occurs when the soil is wet and very high winds blow across the field. In root lodging, the roots are pulled loose from the soil, causing the plant to fall over. Lodging reduces the yield of nonstructural carbohydrates. It is not yet clear which genes show changes in expression when sorghum falls over. We compared whole-gene expression in the mature stems of intact and lodged sorghum plants, with a focus on comparisons from the perspective of differences in sugar accumulation or degradation. Results Lodging decreased sucrose content, starch content, and ratio of sucrose to total sugars in the stems of the sorghum cultivar SIL-05. Particular paralogs of SWEET and TMT family genes, which encode sucrose or hexose transporters, or both, were significantly highly expressed in intact or lodged sorghum stems. In intact stems, genes encoding the glucose-6-phosphate translocator, aquaporins, and enzymes involved in photosynthesis and starch synthesis were highly expressed. In lodged sorghum stems, expression of genes associated with sucrose or starch degradation or energy production was increased. Notably, expression of genes encoding enzymes catalyzing irreversible reactions and associated with the first steps of these metabolic pathways (e.g. INV, SUS, and hexokinase- and fructokinase-encoding genes) was significantly increased by lodging. Expression of SUT, SPS, and SPP was almost the same in intact and lodged sorghum. Conclusions Specific paralogs of sucrose-associated genes involved in metabolic pathways and in membrane transport were expressed in the stems of sorghum SIL-05 at the full-ripe stage. Root lodging drastically changed the expression of these genes from sucrose accumulation to degradation. The changes in gene expression resulted in decreases in sugar content and in the proportion of sucrose to hexoses in the stems of lodged plants.

Upon wounding or pathogen invasion, leaves of sorghum [Sorghum bicolor (L.) Moench] plants with the P gene turn purple, whereas leaves with the recessive allele turn brown or tan. This purple phenotype is determined by the production of two 3-deoxyanthocyanidins, apigeninidin and luteolinidin, which are not produced by the tan-phenotype plants. Using map-based cloning in progeny from a cross between purple Nakei-MS3B (PP) and tan Greenleaf (pp) cultivars, we isolated this gene, which was located in a 27-kb genomic region around the 58.1 Mb position on chromosome 6. Four candidate genes identified in this region were similar to the maize leucoanthocyanidin reductase gene. None of them was expressed before wounding, and only the Sb06g029550 gene was induced in both cultivars after wounding. The Sb06g029550 protein was detected in Nakei-MS3B, but only slightly in Greenleaf, in which it may be unstable because of a Cys252Tyr substitution. A recombinant Sb06g029550 protein had a specific flavanone 4-reductase activity, and converted flavanones (naringenin or eriodictyol) to flavan-4-ols (apiforol or luteoforol) in vitro. Our data indicate that the Sb06g029550 gene is involved in the 3-deoxyanthocyanidin synthesis pathway.

Sorghum (Sorghum bicolor L. Moench) exhibits various color changes in injured leaves in response to cutting stress. Here, we aimed to identify key genes for the light brown and dark brown color variations in tan-colored injured leaves of sorghum. For this purpose, sorghum M36001 (light brown injured leaves), Nakei-MS3B (purple), and a progeny, #7 (dark brown), from Nakei-MS3B × M36001, were used. Accumulated pigments were detected by using high-performance liquid chromatography: M36001 accumulated only apigenin in its light brown leaves; #7 accumulated both luteolin and a small amount of apigenin in its dark brown leaves, and Nakei-MS3B accumulated 3-deoxyanthocyanidins (apigeninidin and luteolinidin) in its purple leaves. Apigenin or luteolin glucoside derivatives were also accumulated, in different proportions. Differentially expressed genes before and after cutting stress were identified by using RNA-seq. Integration of our metabolic and RNA-seq analyses suggested that expression of only flavone synthase II (FNSII) led to the synthesis of apigenin in M36001, expression of both FNSII and flavonoid 3′-hydroxylase (F3′H) led to the synthesis of apigenin and luteolin in #7, and expression of both flavanone 4-reductase and F3’H led to the synthesis of 3-deoxyanthocyanidins in Nakei-MS3B. These results suggest that expression of FNSII is related to the synthesis of flavones (apigenin and luteolin) and the expression level of F3′H is related to the balance of apigenin and luteolin. Expression of FNSII and F3′H is thus associated with dark or light brown coloration in tan-colored injured leaves of sorghum.

The recent development of RNA sequencing (RNA-seq) technology has enabled us to analyze the transcriptomes of plants and their pathogens simultaneously. However, RNA-seq often relies on aligning reads to a reference genome and is thus unsuitable for analyzing most plant pathogens, as their genomes have not been fully sequenced. Here, we analyzed the transcriptomes of Sorghum bicolor (L.) Moench and its pathogen Bipolaris sorghicola simultaneously by using RNA-seq in combination with de novo transcriptome assembly. We sequenced the mixed transcriptome of the disease-resistant sorghum cultivar SIL-05 and B. sorghicola in infected leaves in the early stages of infection (12 and 24 h post-inoculation) by using Illumina mRNA-Seq technology. Sorghum gene expression was quantified by aligning reads to the sorghum reference genome. For B. sorghicola, reads that could not be aligned to the sorghum reference genome were subjected to de novo transcriptome assembly. We identified genes of B. sorghicola for growth of this fungus in sorghum, as well as genes in sorghum for the defense response. The genes of B. sorghicola included those encoding Woronin body major protein, LysM domain-containing intracellular hyphae protein, transcriptional factors CpcA and HacA, and plant cell-wall degrading enzymes. The sorghum genes included those encoding two receptors of the simple eLRR domain protein family, transcription factors that are putative orthologs of OsWRKY45 and OsWRKY28 in rice, and a class III peroxidase that is a homolog involved in disease resistance in the Poaceae. These defense-related genes were particularly strongly induced among paralogs annotated in the sorghum genome. Thus, in the absence of genome sequences for the pathogen, simultaneous transcriptome analysis of plant and pathogen by using de novo assembly was useful for identifying putative key genes in the plant-pathogen interaction.

Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production. [ABSTRACT FROM AUTHOR]

Background: SWEET is a newly identified family of sugar transporters. Although SWEET transporters have been characterized by using Arabidopsis and rice, very little knowledge of sucrose accumulation in the stem region is available, as these model plants accumulate little sucrose in their stems. To elucidate the expression of key SWEET genes involved in sucrose accumulation of sorghum, we performed transcriptome profiling by RNA-seq, categorization using phylogenetic trees, analysis of chromosomal synteny, and comparison of amino acid sequences between SIL-05 (a sweet sorghum) and BTx623 (a grain sorghum). Results: We identified 23 SWEET genes in the sorghum genome. In the leaf, SbSWEET8-1 was highly expressed and was grouped in the same clade as AtSWEET11 and AtSWEET12 that play a role in the efflux of photosynthesized sucrose. The key genes in sucrose synthesis (SPS3) and that in another step of sugar transport (SbSUT1 and SbSUT2) were also highly expressed, suggesting that sucrose is newly synthesized and actively exported from the leaf. In the stem, SbSWEET4-3 was uniquely highly expressed. SbSWEET4-1, SbSWEET4-2, and SbSWEET4-3 were categorized into the same clade, but their tissue specificities were different, suggesting that SbSWEET4-3 is a sugar transporter with specific roles in the stem. We found a putative SWEET4-3 ortholog in the corresponding region of the maize chromosome, but not the rice chromosome, suggesting that SbSWEET4-3 was copied after the branching of sorghum and maize from rice. In the panicle from the heading through to 36 days afterward, SbSWEET2-1 and SbSWEET7-1 were expressed and grouped in the same clade as rice OsSWEET11/Xa13 that is essential for seed development. SbSWEET9-3 was highly expressed in the panicle only just after heading and was grouped into the same clade as AtSWEET8/RPG1 that is essential for pollen viability. Five of 23 SWEET genes had SNPs that caused nonsynonymous amino acid substitutions between SIL-05 and BTx623. Conclusions: We determined the key SWEET genes for technological improvement of sorghum in the production of biofuels: SbSWEET8-1 for efflux of sucrose from the leaf; SbSWEET4-3 for unloading sucrose from the phloem in the stem; SbSWEET2-1 and SbSWEET7-1 for seed development; SbSWEET9-3 for pollen nutrition. [ABSTRACT FROM AUTHOR]

Background: Sorghum (Sorghum bicolor L. Moench) accumulates 3-deoxyanthocyanidins and exhibits orange to purple coloration on parts of the leaf in response to infection with the fungus Bipolaris sorghicola. We aimed to identify the key genes determining this color variation. Results: Sorghum populations derived from Nakei-MS3B and M36001 accumulated apigeninidin, or both apigeninidin and luteolinidin, in different proportions in lesions caused by B. sorghicola infection, suggesting that the relative proportions of the two 3-deoxyanthocyanidins determine color variation. QTL analysis and genomic sequencing indicated that two closely linked loci on chromosome 4, containing the flavonoid 3′-hydroxylase (F3′H) and Tannin1 (Tan1) genes, were responsible for the lesion color variation. The F3′H locus in Nakei-MS3B had a genomic deletion resulting in the fusion of two tandemly arrayed F3′H genes. The recessive allele at the Tan1 locus derived from M36001 had a genomic insertion and encoded a non-functional WD40 repeat transcription factor. Whole-mRNA sequencing revealed that expression of the fused F3′H gene was conspicuously induced in purple sorghum lines. The levels of expression of F3′H matched the relative proportions of apigeninidin and luteolinidin. Conclusions: Expression of F3′H is responsible for the synthesis of luteolinidin; the expression level of this gene is therefore critical in determining color variation in sorghum leaves infected with B. sorghicola. [ABSTRACT FROM AUTHOR]

The spring-type near isogenic line (NIL) of the winter-type barley (Hordeum vulgare ssp. vulgare) var. Hayakiso 2 (HK2) was developed by introducing VERNALIZATION-H1 (Vrn-H1) for spring growth habit from the spring-type var. Indo Omugi. Contrary to expectations, the spring-type NIL flowered later than winter-type HK2. This phenotypic difference was controlled by a single gene, which cosegregated only with phytochrome C (HvPhyC) among three candidates around the Vrn-H1 region (Vrn-H1, HvPhyC, and CASEIN KINASE IIα), indicating that HvPhyC was the most likely candidate gene. Compared with the late-flowering allele HvPhyC-l from the NIL, the early-flowering allele HvPhyC-e from HK2 had a single nucleotide polymorphism T1139C in exon 1, which caused a nonsynonymous amino acid substitution of phenylalanine at position 380 by serine in the functionally essential GAF (3', 5'-cyclic-GMP phosphodiesterase, adenylate cyclase, formate hydrogen lyase activator protein) domain. Functional assay using a rice (Oryza sativa) phyA phyC double mutant line showed that both of the HvPhyC alleles are functional, but HvPhyC-e may have a hyperfunction. Expression analysis using NILs carrying HvPhyC-e and HvPhyC-l (NIL [HvPhyC-e] and NIL [HvPhyC-l], respectively) showed that HvPhyC-e up-regulated only the flowering promoter FLOWERING LOCUS T1 by bypassing the circadian clock genes and flowering integrator CONSTANS1 under a long photoperiod. Consistent with the up-regulation, NIL (HvPhyC-e) flowered earlier than NIL (HvPhyC-l) under long photoperiods. These results implied that HvPhyC is a key factor to control long-day flowering directly. [ABSTRACT FROM AUTHOR]

Abstract  Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin. [ABSTRACT FROM AUTHOR]

We introduced two novel types of pig (Sus scrofa) cytochrome P450, CYP2B22 and CYP2C49, into rice plants (Oryza sativa L. cv. ‘Nipponbare’) to produce herbicide-tolerant plants and to confirm the metabolic activities of the cytochrome P450 species. In germination tests, both types of transgenic plants showed tolerance to various herbicides with different modes of action. CYP2B22 rice plants showed tolerance towards 12 herbicides including chlortoluron (100 μM), amiprofos-methyl (2.5 μM), pendimethalin (10 μM), metolachlor (2.5 μM), and esprocarb (20 μM). CYP2C49 rice plants showed tolerance towards 13 herbicides, including chlortoluron (100 μM), norflurazon (0.5 μM), amiprofos-methyl (2.5 μM), alachlor (0.8 μM), and isoxaben (1 μM). The herbicide tolerance was considered to reflect the substrate specificity of the introduced P450 species. We used 14C-labeled metolachlor and norflurazon to confirm the P450 activity in the transgenic rice plants. The herbicides were metabolized more quickly in the transgenic rice plants than in the nontransgenic rice plants. Therefore, CYP2B22 and CYP2C49 rice plants became more tolerant to various herbicides than nontransgenic control plants because of accelerated metabolism of the herbicides by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, these transgenic rice plants may become useful tools for the breeding of herbicide-tolerant crops. [ABSTRACT FROM AUTHOR]

To clarify the genetic mechanisms of fertility restoration in sorghum F¹ hybrids produced in Japan ('Ryokuryu', 'Hazuki', 'Haretaka', 'Natsuibuki', 'Hanaaoba', 'Akidachi' and 'Kazetachi'), we analyzed QTLs for fertility restoration using seven F2 populations derived from those hybrids. By QTL mapping with a series of SSR markers, we detected three major QTLs for fertility restoration. These data and the results of haplotype analysis of known fertility restorer (Rf) genes showed that qRf5, corresponding to the Rf5 locus, was the most widely used Rf gene for fertility restoration of sorghum F¹ hybrids among the lines tested. Other major Rf genes detected were qRf8, corresponding to Rf¹, and qRf2, corresponding to Rf2. QTLs for grain weight also corresponded to these Rf loci. A minor QTL, qRf3, may also affect restoration of fertility. Our data show that three major Rfs--Rf1, Rf2, and Rf5--were used in F¹ hybrid sorghum production in Japan. This knowledge can be used to improve the efficiency of the F¹ sorghum breeding program. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility (CMS) is widely used to control pollination in the production of commercial F1 hybrid seed in sorghum. So far, 6 major fertility restorer genes, Rf1 to Rf6, have been reported in sorghum. Here, we fine-mapped the Rf5 locus on sorghum chromosome 5 using descendant populations of a ‘Nakei MS-3A’ × ‘JN43’ cross. The Rf5 locus was narrowed to a 140-kb region in BTx623 genome (161-kb in JN43) with 16 predicted genes, including 6 homologous to the rice fertility restorer Rf1 (PPR.1 to PPR.6). These 6 homologs have tandem pentatricopeptide repeat (PPR) motifs. Many Rf genes encode PPR proteins, which bind RNA transcripts and modulate gene expression at the RNA level. No PPR genes were detected at the Rf5 locus on the corresponding homologous chromosome of rice, foxtail millet, or maize, so this gene cluster may have originated by chromosome translocation and duplication after the divergence of sorghum from these species. Comparison of the sequences of these genes between fertile and CMS lines identified PPR.4 as the most plausible candidate gene for Rf5. [ABSTRACT FROM AUTHOR]