Depending on what type, they may carry starch, proteins, fats, pigments, and even crystals. F2 plants (n = 222) had a 3:1 ratio of dry (n = 168, 75.7%) to juicy (n = 54, 24.3%) stem phenotypes, suggesting that the dry stem with dead pith parenchyma cells is a dominant trait, whereas the juicy stem with live pith parenchyma cells is a recessive trait. D expression was lowest in the first and seventh internodes (Fig. Thus, our study revealed the molecular and cellular mechanism determining stem water content in sorghum and possibly other grasses, crops, and vegetables. Phenotypes of dry- and juicy-stem sorghum varieties. 2B). Consistent with this, most of the studied VNS members activate genes related to cell-wall formation and PCD (26, 31, 37). Author contributions: M.F., J.-i. 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. Fine mapping with 1,925 F2 individuals and the SNP/InDel markers confined the D locus to an 18.99-kb region (from 51.794 to 51.808 Mb) on chromosome 6 (Fig. Exon sequence analysis of 93 germplasms with various stem juice sugar concentrations identified three additional nonfunctional alleles in the juicy-stem varieties (Dataset S1). HIS3 and ADE2 genes under control of GAL4-binding sites serve as reporters. Relative LUC activity in protoplasts transfected with the effector construct harboring CaMV35Spro::::CaMV35Ster (the only regulatory elements) and the reporter construct harboring CEP1pro(−555)::LUC::CaMV35Ster was defined as 1.0. Thus, among these functionally differentiated group II NAC subfamily proteins, so far, only D subfamily proteins—at least those in sorghum, Arabidopsis, and rice—have been shown to have the ability to induce PCD. The other experiment was a yeast one-hybrid assay for CEP1, which showed that both D and ANAC074 specifically bound to the CEP1 5′-upstream region (SI Appendix, Fig. This tissue is “functional” – performing tasks such as photosynthesis in plants or storing information in the human brain – as opposed to “structural” tissues like wood in plants or bone in animals. D expression was much lower in the second, fifth, and sixth internodes (∼19, 19, and 3 times higher, respectively, than that in the first internode of 74LH3213) (Fig. In sweet sorghum varieties, as in sugarcane, the sugar yield increases with increasing stem water content and sugar concentration, both of which are targets of breeding programs (11). 5A, arrowheads). S1), as described previously (15, 17). NAC1 encodes a NAC protein belonging to the NAC1 subfamily, which is phylogenetically close to, but distinct from, the D subfamily (SI Appendix, Fig. This is consistent with another study that classified plant NAC superfamily proteins into six groups (25), in which groups I and II corresponded to VNS and D subfamily proteins, respectively. 5B, Upper). Analysis of the SKS and MS3B BAC clones and reference genome sequences corresponding to the 18.99-kb region identified only one gene, Sobic.006G147400 (Fig. Storage of food 4. S3A and Dataset S1). Parenchyma is the tissue made up of cells and intercellular spaces that fills the interior of the body of a flatworm, which is an acoelomate. The preparation has been stained with coriphosphin. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production. Most photosynthesis occurs in the leaves of a plant, which contain numerous parenchyma cells with chloroplasts. The inclusions in xylem parenchyma cells may contain tanninferous compounds. The difference in stem water contents between SKS and MS3B is readily observed by squeezing the stems (Movie S1). contributed new reagents/analytic tools; M.F., Y.O., H.K., M.I., H.K.-K., H.I., and J.-i. (F) Cross-sections of the second stem internodes of 74LH3213 at 7–11 WAP. From the Cambridge English Corpus The … D was specifically expressed in 74LH3213 stems but not in d-NIL stems (Fig. Plant Cell and Environment 40: 858–871. Ø They are the parenchyma cells occur in the vascular tissue of plants. 3C), where the greatest formation of white pith parenchyma was observed (Fig. 5C). 3B). (F) Hoechst 33342-stained vertical sections of the third internodes in SKS and MS3B stems at 9 WAP. 4D). are listed on a patent based on this work (Japanese patent P2015-025241), which was filed by NARO. The gene responsible for determining dry-stem and juicy-stem traits of sorghum has long been referred to as D. In this study, we identified an uncharacterized gene, Sobic.006G147400, as a candidate for D. We could not identify any other genes within the genomic region for D determined by our fine mapping. Therefore, we used Arabidopsis for these studies because it is easily transformable and has a single D homolog, ANAC074 (SI Appendix, Fig. 6C). 3C). This has led us to speculate that D may be involved in determining the death of pith parenchyma cells in sorghum stems. 4C), providing further evidence that ANAC074 is expressed in these cells. 1 F and G), confirming that the latter were dead. Article Google Scholar Trifilo P., P. M. … (Scale bars: 10 µm in A.). PCD in pith parenchyma of flowering plant stems has been proposed to reduce stem water content (4), facilitate nutrient translocation from stems to sink organs (3, 4), and provide drought or waterlogging stress tolerance (5⇓–7). This is a spongy tissue also known as a mesenchymal tissue, in which several types of cells are lodged in their extracellular matrices. Induction of D or ANAC074 also up-regulated the expression of other genes encoding autolytic enzymes, which generally function to execute PCD, including an aspartic endopeptidase (PASPA3), a serine carboxypeptidase-like acyltransferase (SCPL48), a bifunctional nuclease (BFN1), and a ribonuclease (RNS3) (29) (Fig. These results show that SKS and MS3B stems are well suited for studies of the D gene, which determines the dry- or juicy-stem trait. First, in our phylogenic tree (SI Appendix, Fig. The palisade parenchyma is interpreted as a barrier to desiccation of the acorn and may have a photosynthetic function during the formation of the fruit. Thank you for your interest in spreading the word on PNAS. S8, dark ocher squares), suggesting that these cells had undergone PCD. D complements mutant anac074 plant phenotypes and induces ectopic PCD in Arabidopsis culture cells by up-regulating the Arabidopsis PCD-executing enzymes. We thank N.-H. Chua (Rockefeller University), U. Grossniklaus (University of Zurich), and The Nottingham Arabidopsis Stock Centre for providing the pER8 vector, the pER8-modified pMDC7 vector, and the Arabidopsis mutant (GK_224H04), respectively; T. Ando, T. Mizubayashi, H. Kanamori, S. H. Choi (NARO), Y. Hirata, S. Lin, T. Hidaka, Y. Sano, M. Ueda, K. Bou, and Y. Tamura (The University of Tokyo) for technical assistance; and T. Fujiwara and M. Tanaka (The University of Tokyo) for assistance with the luciferase-based transactivation assay. para, “beside” + enchyma, “an infusion”) make up one of the three ground tissues, alongside collenchyma and sclerenchyma cells. All images were acquired at 48 h after the addition of estrogen. Some NAC transcription factors, such as the Arabidopsis VNS subfamily protein VND6 (SI Appendix, Fig. Some VNS subfamily proteins, like some D subfamily proteins, function as master switches for PCD (26, 31, 32). D and its Arabidopsis ortholog encode master transcriptional switches that induce programmed death of stem pith parenchyma cells by activating autolytic enzymes. S10). Yonemaru performed research; T.S., Y.O., K.E., T.U., and T.T. Color scale indicates fold-changes in gene expression level (on a log-2 scale). Insets display magnified images of a vascular bundle. Different letters indicate statistically significant differences (Tukey’s honestly significant difference; α = 0.05). The expression level of ANAC074 in 65-d-old inflorescence stems was significantly higher than the expression levels in 10-d-old seedlings, young and mature rosette leaves, flower buds, flowers, 50- and 65-d-old hypocotyls, and 35- and 50-d-old inflorescence stems (Fig. Error bars show SD (n = 3). This strongly suggests that Sobic.006G147400 is the D gene that determines stem water content in sorghum. These results indicate that the functions of sorghum D are compatible with those of Arabidopsis ANAC074. We demonstrated that ANAC074, an Arabidopsis ortholog of D, was preferentially expressed in flowers, older hypocotyls, and older inflorescence stems and that ANAC074 is necessary and sufficient to induce cell death, at least in pith parenchyma of older inflorescence stems. Different letters indicate statistically significant differences (Tukey’s honestly significant difference; α = 0.05). Thin walls allow for close packing and rapid diffusion between cells. 4D), confirming the involvement of D in cell death of stem pith parenchyma. S5B), and xylem and degenerating pith parenchyma of 65-d-old inflorescence stems (Fig. S4). (Lower) Bar graphs represent the relative activities of firefly LUC in Arabidopsis culture cell protoplasts transfected with the indicated combinations of the effector and reporter constructs. In Arabidopsis culture cells, GFP-fused D or GFP-fused ANAC074 localized to the nucleus before cell death (Fig. 5 A–C and SI Appendix, Fig. (C) Gene structures of D alleles in 13 sorghum cultivars. Most of the SKS pith parenchyma cells are filled with air bubbles, whereas the MS3B pith parenchyma cells retain their cellular contents (Fig. (A) Stem juice content in Senkinshiro (SKS, dry-stem variety) and Nakei-MS3B (MS3B, juicy-stem variety) at 30 DAH. (Scale bars: 5 mm and 100 µm in Upper and Lower, respectively, of D; 100 µm in F; and 2 mm and 50 μm in Lower Right and Upper Right, respectively, in H.). Additional mapping of 1,000 F2 individuals narrowed the D locus to a 185-kb region (from 51.788 to 51.973 Mb) on chromosome 6 (Fig. Error bars show SD (n = 3). In situ hybridization analysis of 74LH3213 stems at 8 WAP showed that D was expressed in pith parenchyma cells within the second internode portion just before the appearance of white pith parenchyma (Fig. These results suggest that at least three D subfamily members, sorghum D, Arabidopsis ANAC074, and rice OsD, share overlapping functions involved in cell death. Characteristics . To characterize the cellular function of D, we compared the effects of ectopic expression of D, ANAC074, OsD, and Arabidopsis NAC1 in Arabidopsis culture cells. Annual Review of Phytopathology 22: 189–214. Genome editing technologies can be used to modulate the activity of endogenous D in breeding programs for a range of crops and vegetables. S3B). Hereafter, we refer to Sobic.006G147400 as D. To determine the historical origin of juicy-stem varieties with nonfunctional D alleles, we compared the distribution of functional and nonfunctional D alleles in a geographical origin analysis of 93 germplasms from Asia and Africa. To confirm that D and ANAC074 directly target PCD-related genes, we performed two experiments using the 5′-upstream region of CEP1, a gene that encodes a cysteine peptidase and is up-regulated by expression of D or ANAC074 in Arabidopsis culture cells (Fig. In 75-d-old senescent inflorescence stems of wild-type plants, most pith parenchyma cells were stained by Evans blue (Fig. These results indicated that functional and nonfunctional D alleles were widely distributed in Asian and African germplasms (SI Appendix, Fig. D expression in each internode of d-NIL stems was not detected or was much lower than D expression in 74LH3213 stems (Fig. 2C and SI Appendix, Fig. Therefore, juicy- and dry-stem traits of sorghum are tightly coupled with the abundance of living and dead pith parenchyma cells, respectively. Details for plant materials, calculation of stem juice content, measurement of sugar concentration (Brix), calculation of stem sugar content, air porosity measurement, map-based cloning, phylogenetic analysis, polymorphism analysis, quantitative RT-PCR, plasmid construction, analysis of amino acid sequence homology, Arabidopsis transformation, in situ hybridization, histochemical GUS staining, microscopic observations, transcriptional activation assay in yeast cells, luciferase-based transactivation assay in Arabidopsis protoplasts, yeast one-hybrid assay, 3′-tag digital gene expression profiling, gene ontology analysis, statistical analysis, and data availability are described in SI Appendix, Materials and Methods. SOKENDAI (The Graduate University for Advanced Studies, Japan Science and Technology Agency (JST), From thin to thick: Major transitions during stem development, Programmed cell death during plant growth and development, Autolysis in herbaceous, dicotyledonous plants: Experimental manipulation of pith autolysis in several cultivated species, Non-structural carbohydrate partitioning in grass stems: A target to increase yield stability, stress tolerance, and biofuel production, Stem pithiness in tomato plants: The effect of water-stress and the role of abscisic-acid, Role of cellulase in aerenchyma development in sunflower, Aerenchyma formation in the rice stem and its promotion by H, General and specific combining ability estimates for pith cell-death in stalk internodes of maize, Sorghum: Origin, History, Technology and Production, Genetics, Genomics and Breeding of Sorghum, QTLs for energy-related traits in a sweet x grain sorghum [, On the inheritance of certain stem characters in sorghum, Inheritance of smut resistance and juiciness of stalk in the sorghum cross, red amber X feterita, Location of major effect genes in sorghum (, Identification of quantitative trait loci for agronomically important traits and their association with genic-microsatellite markers in sorghum, Genomewide association for sugar yield in sweet sorghum, Combining next generation sequencing with bulked segregant analysis to fine map a stem moisture locus in sorghum (, DNA-binding domains of plant-specific transcription factors: Structure, function, and evolution, NAC transcription factors: Structurally distinct, functionally diverse, Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development, Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants, Comparative genomics of NAC transcriptional factors in angiosperms: Implications for the adaptation and diversification of flowering plants, Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 directly regulates the genes that govern programmed cell death and secondary wall formation during xylem differentiation, Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein, Only in dying, life: Programmed cell death during plant development, A conserved core of programmed cell death indicator genes discriminates developmentally and environmentally induced programmed cell death in plants, Morphological classification of plant cell deaths, Contribution of NAC transcription factors to plant adaptation to land, Transcription switches for protoxylem and metaxylem vessel formation, The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation, The CUP-SHAPED COTYLEDON3 gene is required for boundary and shoot meristem formation in Arabidopsis, EIN3 and ORE1 accelerate degreening during ethylene-mediated leaf senescence by directly activating chlorophyll catabolic genes in Arabidopsis, NAC transcription factor ORE1 and senescence-induced BIFUNCTIONAL NUCLEASE1 (BFN1) constitute a regulatory cascade in Arabidopsis, VASCULAR-RELATED NAC-DOMAIN7 directly regulates the expression of a broad range of genes for xylem vessel formation, Mutation of WRKY transcription factors initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants, Composition of cell walls isolated from cell types of grain sorghum stems, The genetic control of lignin deposition during plant growth and development, Seasonal trends in density and cell death in sorghum stalk tissue, Relationship of cell death patterns and spread of, Breeding for lodging resistance in sorghum, Proceedings of the 32nd Annual Corn and Sorghum Industry Research Conference, Breeding for resistance to root and stalk rots in Texas, Sorghum Root and Stalk Rots: A Critical Review, Proceedings of the National Academy of Sciences, Earth, Atmospheric, and Planetary Sciences, https://www.ddbj.nig.ac.jp/dra/index.html, www.pnas.org/lookup/suppl/doi:10.1073/pnas.1807501115/-/DCSupplemental, News Feature: To understand the plight of insects, entomologists look to the past, Opinion: We need to improve the welfare of life science trainees, Journal Club: Clues to Alzheimer’s disease onset in the aging female brain, Protecting against spaceflight-induced muscle and bone loss. 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To get trusted stories delivered Right to your inbox, does not induce ectopic PCD in Arabidopsis culture cell.... In plants parenchyma cells and aquaporins during recovery from severe water stress type is present, which phylogenetically...