The tuber-specific StbHLH93 gene regulates proplastid-to-amyloplast development during stolon swelling in potato.

In potato, stolon swelling is a complex and highly regulated process, and much more work is needed to fully understand the underlying mechanisms. We identified a novel tuber-specific basic helix-loop-helix (bHLH) transcription factor, StbHLH93, based on the high-resolution transcriptome of potato tuber development. StbHLH93 is predominantly expressed in the subapical and perimedullary region of the stolon and developing tubers. Knockdown of StbHLH93 significantly decreased tuber number and size, resulting from suppression of stolon swelling. Furthermore, we found that StbHLH93 directly binds to the plastid protein import system gene TIC56 promoter, activates its expression, and is involved in proplastid-to-amyloplast development during the stolon-to-tuber transition. Knockdown of the target TIC56 gene resulted in similarly problematic amyloplast biogenesis and tuberization. Taken together, StbHLH93 functions in the differentiation of proplastids to regulate stolon swelling. This study highlights the critical role of proplastid-to-amyloplast interconversion during potato tuberization.

Fine-scale clonal structure of the lingonberry Vaccinium vitis-idaea under the nurse plant Pinus pumila vegetation in an alpine region, Mt. Norikura.

The nurse effect is a positive interaction in which a nurse plant improves the abiotic environment for another species (beneficiary plant) and facilitates its establishment. The evergreen shrub Vaccinium vitis-idaea (a beneficiary plant) grows mainly under the dwarf shrub Pinus pumila (a nurse plant) in the alpine regions of central Japan. However, whether V. vitis-idaea shrubs under various P. pumila shrubs spread through clonal growth and/or seeds remains unclear. We investigated the clonal structure of V. vitis-idaea under the nurse plant P. pumila in Japanese alpine regions. MIG-seq analysis was conducted to clarify the clonal diversity of V. vitis-idaea in isolated and patchy P. pumila plots on a ridge (PATs), and in a plot covered by dense P. pumila on a slope adjacent to the ridge (MAT) on Mt. Norikura, Japan. We detected 28 multilocus genotypes in 319 ramets of V. vitis-idaea across 11 PATs and MAT. Three genets expanded to more than 10 m in the MAT. Some genets were shared among neighboring PATs or among PATs and MAT. These findings suggest that the clonal growth of V. vitis-idaea plays an important role in the sustainability of populations. The clonal diversity of V. vitis-idaea was positively related with the spatial size of PATs and was higher in MAT than in PATs at a small scale. Therefore, the spatial spread of the nurse plant P. pumila might facilitate the nurse effect on the genetic diversity of beneficiary plants, leading to the sustainability of beneficiary populations.

Proteome-wide analyses reveal diverse functions of protein acetylation and succinylation modifications in fast growing stolons of bermudagrass (Cynodon dactylon L.).

BACKGROUND: Bermudagrass (Cynodon dactylon L.) is an important warm-season turfgrass species with well-developed stolons, which lay the foundation for the fast propagation of bermudagrass plants through asexual clonal growth. However, the growth and development of bermudagrass stolons are still poorly understood at the molecular level. RESULTS: In this study, we comprehensively analyzed the acetylation and succinylation modifications of proteins in fast-growing stolons of the bermudagrass cultivar Yangjiang. A total of 4657 lysine acetylation sites on 1914 proteins and 226 lysine succinylation sites on 128 proteins were successfully identified using liquid chromatography coupled to tandem mass spectrometry, respectively. Furthermore, 78 proteins and 81 lysine sites were found to be both acetylated and succinylated. Functional enrichment analysis revealed that acetylated proteins regulate diverse reactions of carbohydrate metabolism and protein turnover, whereas succinylated proteins mainly regulate the citrate cycle. These results partly explained the different growth disturbances of bermudagrass stolons under treatment with sodium butyrate and sodium malonate, which interfere with protein acetylation and succinylation, respectively. Moreover, 140 acetylated proteins and 42 succinylated proteins were further characterized having similarly modified orthologs in other grass species. Site-specific mutations combined with enzymatic activity assays indicated that the conserved acetylation of catalase and succinylation of malate dehydrogenase both inhibited their activities, further implying important regulatory roles of the two modifications. CONCLUSION: In summary, our study implied that lysine acetylation and succinylation of proteins possibly play important regulatory roles in the fast growth of bermudagrass stolons. The results not only provide new insights into clonal growth of bermudagrass but also offer a rich resource for functional analyses of protein lysine acetylation and succinylation in plants.

Comparative proteomic analysis provides new insights into the specialization of shoots and stolons in bermudagrass (Cynodon dactylon L.).

BACKGROUND: Bermudagrass (Cynodon dactylon L.) is an important turfgrass species with two types of stems, shoots and stolons. Despite their importance in determining the morphological variance and plasticity of bermudagrass, the intrinsic differences between stolons and shoots are poorly understood. RESULTS: In this study, we compared the proteomes of internode sections of shoots and stolons in the bermudagrass cultivar Yangjiang. The results indicated that 376 protein species were differentially accumulated in the two types of stems. Pathway enrichment analysis revealed that five and nine biochemical pathways were significantly enriched in stolons and shoots, respectively. Specifically, enzymes participating in starch synthesis all preferentially accumulated in stolons, whereas proteins involved in glycolysis and diverse transport processes showed relatively higher abundance in shoots. ADP-glucose pyrophosphorylase (AGPase) and pyruvate kinase (PK), which catalyze rate-limiting steps of starch synthesis and glycolysis, showed high expression levels and enzyme activity in stolons and shoots, respectively, in accordance with the different starch and soluble sugar contents of the two types of stems. CONCLUSIONS: Our study revealed the differences between the shoots and stolons of bermudagrass at the proteome level. The results not only expand our understanding of the specialization of stolons and shoots but also provide clues for the breeding of bermudagrass and other turfgrasses with different plant architectures.

Identification and characterization of proteins involved in stolon adhesion in the highly invasive fouling ascidian Ciona robusta.

Adhesive ascidians have caused serious biofouling problems and huge economic losses in marine ecosystems. However, adhesion mechanisms, particularly on functional proteins involved in ascidian adhesion, remain largely unexplored. Here, we identified 26 representative stolon proteins from the highly invasive fouling ascidian Ciona robusta using the proteomics approach. The uncharacterized stolon proteins were rich in adhesion-related conserved domains. Real-time quantitative PCR further revealed specific expressions of these uncharacterized protein genes in stolon tissue, suggesting their potential roles in stolon adhesion.> A recombinant vWFA domain-containing uncharacterized protein, ascidian stolon protein 1 (ASP-1), was successfully expressed in a baculovirus-insect cell system and purified in vitro. Coating experiment showed that tyrosinase-modified ASP-1 could absorb to glass and organic glass stronger than unmodified ASP-1, while only modified ASP-1 could absorb to aluminum foil. Quartz crystal microbalance analysis also showed the increase in absorption ability of ASP-1 after modification. In addition, abundant 3,4-l-dihydroxyphenylalanine (DOPA) in modified protein was detected by nitroblue tetrazolium staining. These results suggest that ASP-1 be involved in ascidian DOPA-dependent and material-selective adhesion. Overall, this study provides insight into molecular mechanisms of C. robusta stolon adhesion, and findings here are expected to be conductive to develop strategies against biofouling caused by ascidians.

Phenotypic analysis combined with tandem mass tags (TMT) labeling reveal the heterogeneity of strawberry stolon buds.

BACKGROUND: Ramet propagation in strawberry (Fragaria × ananassa) is the most effective way in production. However, the lack of systematically phenotypic observations and high-throughput methods limits our ability to analyze the key factors regulating the heterogeneity in strawberry stolon buds. RESULTS: From observation, we found that the axillary bud located in the first node quickly stepped into dormancy (DSB), after several bract and leaf buds were differentiated. The stolon apical meristem (SAM) degenerated as the new ramet leaf buds (RLB), and the new active axillary stolon buds (ASB) differentiated continually after the differentiation of the first leaf. Using the tandem mass tags (TMT) labeling method, a total of 7271 strawberry proteins were identified. Between ASB and DSB, the spliceosome DEPs, such as Ser/Arg-rich (SR) and heterogeneous nuclear ribonucleoprotein particle (hnRNP), showed the highest enrichment and high PPI connectivity. This indicated that the differences in DEPs (e.g., SF-3A and PK) at the transcriptional level may be causing the differences between the physiological statuses of ASB and DSB. As expected, the photosynthetic pre-form RLB mainly differentiated from ASB and DSB judging by the DEP enrichment of photosynthesis. However, there are still other specialized features of DEPs between RLB and DSB and between ASB and DSB. The DEPs relative to DNA duplication [e.g., minichromosome maintenance protein (MCM 2, 3, 4, 7)], provide a strong hint of functional gene duplication leading the bud heterogeneity between RLB and DSB. In addition, the top fold change DEP of LSH 10-like might be involved in the degeneration of SAM into RLBs, based on its significant function in modulating the plant shoot initiation. As for RLB/ASB, the phenylpropanoid biosynthesis pathway probably regulates the ramet axillary bud specialization, and further promotes the differentiation of xylem when ASB develops into a new stolon [e.g., cinnamyl alcohol dehydrogenase 1 (CAD1) and phenylalanine ammonia-lyase 1 (PAL1)]. CONCLUSIONS: By using phenotypic observation combined with proteomic networks with different types of strawberry stolon buds, the definite dormancy phase of DSB was identified, and the biological pathways and gene networks that might be responsible for heterogeneity among different stolon buds in strawberry were also revealed.

Genome-wide transcriptome analysis reveals small RNA profiles involved in early stages of stolon-to-tuber transitions in potato under photoperiodic conditions.

BACKGROUND: Small RNAs (sRNAs), especially miRNAs, act as crucial regulators of plant growth and development. Two other sRNA groups, trans-acting short-interfering RNAs (tasiRNAs) or phased siRNAs (phasiRNAs), are also emerging as potential regulators of plant development. Stolon-to-tuber transition in potato is an important developmental phase governed by many environmental, biochemical and hormonal cues. Among different environmental factors, photoperiod has a major influence on tuberization. Several mobile signals, mRNAs, proteins and transcription factors have been widely studied for their role in tuber formation in potato, however, no information is yet available that describes the molecular signals governing the early stages of stolon transitions or cell-fate changes at the stolon tip before it matures to potato. Stolon could be an interesting model for studying below ground organ development and we hypothesize that small RNAs might be involved in regulation of stolon-to-tuber transition process in potato. Also, there is no literature that describes the phased siRNAs in potato development. RESULTS: We performed sRNA profiling of early stolon stages (4, 7 and 10 d) under long-day (LD; 16 h light, 8 h dark) and short-day (SD; 8 h light, 16 h dark) photoperiodic conditions. Altogether, 7 (out of 324) conserved and 12 (out of 311) novel miRNAs showed differential expression in early stolon stages under SD vs LD photoperiodic conditions. Key target genes (StGRAS, StTCP2/4 and StPTB6) exhibited differential expression in early stolon stages under SD vs LD photoperiodic conditions, indicative of their potential role in tuberization. Out of 830 TAS-like loci identified, 24 were cleaved by miRNAs to generate 190 phased siRNAs. Some of them targeted crucial tuberization genes such as StPTB1, POTH1 and StCDPKs. Two conserved TAS loci, referred as StTAS3 and StTAS5, which share close conservation with members of the Solanaceae family, were identified in our analysis. One TAS-like locus (StTm2) was validated for phased siRNA generation and one of its siRNA was predicted to cleave an important tuber marker gene StGA2ox1. CONCLUSION: Our study suggests that sRNAs and their selective target genes could be associated with the regulation of early stages of stolon-to-tuber transitions in a photoperiod-dependent manner in potato.

Large contribution of clonal reproduction to the distribution of deciduous liana species (Wisteria floribunda) in an old-growth cool temperate forest: evidence from genetic analysis.

Background and Aims: Extensive clonal (vegetative) reproduction in lianas is a common and important life history strategy for regeneration and colonization success. However, few studies have evaluated the contribution of clonal reproduction to stand-level distribution of lianas in their natural habitat using genetic tools. The objectives of the present study were to investigate (1) the contribution of clonal reproduction to the distribution of Wisteria floribunda, (2) the size of clonal patches and (3) how the distribution patterns of W. floribunda clones are affected by micro-topography. Methods: The contribution of clonal reproduction to the distribution of the deciduous liana species W. floribunda was evaluated using genetic analysis across a 6-ha plot of an old-growth temperate forest in Japan and preference in landform between clonal ramets and non-clonal ramets was assessed. Key Results: Of the 391 ramets sampled, clonal reproduction contributed to 71 and 62 % of the total abundance and basal area, respectively, or 57 and 31 % when the largest ramet within a genet was excluded. The large contribution of clonal reproduction to the density and basal area of W. floribunda was consistent with previous observational studies. The largest genet included a patch size of 0.47 ha and ranged over 180 m. Preferred landforms of clonal and non-clonal ramets were significantly different when evaluated by both abundance and basal area. Non-clonal ramets distributed more on lower part of the slope than other landforms in comparison with clonal ramets and trees, possibly reflecting the limitation of clonal growth by stolons. Conclusions: Using genetic analysis, the present study found evidence of a large contribution of clonal reproduction on the distribution of W. floribunda in its natural habitat. The results indicate that clonal reproduction plays an important role not only in the formation of populations but also in determining the distribution patterns of liana species.

Transgenic creeping bentgrass plants expressing a Picea wilsonii dehydrin gene (PicW) demonstrate improved freezing tolerance.

Agrostis stolonifera L. 'Penn A-4' is a common creeping bentgrass species that is widely used in urban landscaping and golf courses. To prolong the green stage of this grass, a dehydrin gene PicW isolated from Wilson's spruce (Picea wilsonii) was transformed into plants of 'Penn A-4' cultivar via a straightforward stolon node infection system. A putative transgenic plant was obtained and its tolerance to low-temperature stress was evaluated. When the transgenic line was subjected to a freezing (- 5 °C) treatment, it showed better viability and more robust physiology than wild type, as evidenced by higher soluble sugar and proline contents, and lower relative electrical conductivity and malondialdehyde content. The transgenic line also showed tolerance to a chilling treatment (5 °C), although its performance was not significantly different from that of wild-type plants. Overall, the research here clearly revealed the explicit role of PicW in increasing freezing tolerance of grass at the whole-plant level, and demonstrated that the straightforward stolon node transformation method could be well used to genetically modify turfgrass. The obtained transgenic line might be as genetic resource for breeding program and practiced to grow in cold temperate zones.

The plasma membrane H+-ATPase gene family in Solanum tuberosum L. Role of PHA1 in tuberization.

This study presents the characterization of the plasma membrane (PM) H+-ATPases in potato, focusing on their role in stolon and tuber development. Seven PM H+-ATPase genes were identified in the Solanum tuberosum genome, designated PHA1-PHA7. PHA genes show distinct expression patterns in different plant tissues and under different stress treatments. Application of PM H+-ATPase inhibitors arrests stolon growth, promotes tuber induction, and reduces tuber size, indicating that PM H+-ATPases are involved in tuberization, acting at different stages of the process. Transgenic potato plants overexpressing PHA1 were generated (PHA1-OE). At early developmental stages, PHA1-OE stolons elongate faster and show longer epidermal cells than wild-type stolons; this accelerated growth is accompanied by higher cell wall invertase activity, lower starch content, and higher expression of the sucrose-H+ symporter gene StSUT1. PHA1-OE stolons display an increased branching phenotype and develop larger tubers. PHA1-OE plants are taller and also present a highly branched phenotype. These results reveal a prominent role for PHA1 in plant growth and development. Regarding tuberization, PHA1 promotes stolon elongation at early stages, and tuber growth later on. PHA1 is involved in the sucrose-starch metabolism in stolons, possibly providing the driving force for sugar transporters to maintain the apoplastic sucrose transport during elongation.

On the role of the proventricle region in reproduction and regeneration in Typosyllis antoni (Annelida: Syllidae).

BACKGROUND: Syllids are a species rich annelid family possessing remarkable regenerative ability, which is not only the response after traumatic injury, but also a key step during the life cycle of several syllid taxa. In these animals the posterior part of the body becomes an epitoke and is later detached as a distinct unit named stolon. Such a sexual reproductive mode is named schizogamy or stolonization. The prostomium and the proventricle, a modified foregut structure, have been proposed to have a control function during this process, though the concrete mechanisms behind it have never been elucidated. RESULTS: By using different experimental set-ups, histology and immunohistochemistry combined with subsequent cLSM analyzes, we investigate and document the regeneration and stolonization in specimens of Typosyllis antoni that were amputated at different levels throughout the antero-posterior body axis. The removal of the anterior end including the proventricle implies an incomplete anterior regeneration as well as severe deviations from the usual reproductive pattern, i.e. accelerated stolonization, masculinization and the occurrence of aberrant stolons. The detailed anatomy of aberrant stolons is described. A histological study of the proventricle revealed no signs of glandular or secretory structures. The ventricle and the caeca are composed of glandular tissue but they are not involved in the reproductive and regenerative processes. CONCLUSIONS: As in other investigated syllids, the proventricle region has a significant role during stolonization and reproduction processes in Typosyllis antoni. When the proventricle region is absent, anterior and posterior regeneration are considerably deviated from the general patterns. However, proventricle ultrastructure does not show any glandular component, thereby questioning a direct involvement of this organ itself in the control of reproduction and regeneration. Our findings offer a comprehensive starting point for further studies of regeneration and reproductive control in syllids as well as annelids in general.

Potato yield enhancement through intensification of sink and source performances.

The combined total annual yield of six major crops (maize, rice, wheat, cassava, soybean, and potato; Solanum tuberosum L.) amounts to 3.1 billion tons. In recent years, staple crops have begun to be used as substitutes for fossil fuel and feedstocks. The diversion of crop products to fuels and industrial feedstocks has become a concern in many countries because of competition for arable lands and increased food prices. These concerns are definitely justified; however, if plant biotechnology succeeds in increasing crop yields to double the current yields, it will be possible to divert the surplus to purposes other than food without detrimental effects. Maize, rice, wheat, and soybean bear their sink organs in the aerial parts of the plant, and potato in the underground parts. Plants with aerial storage organs cannot accumulate products beyond their capacity to support the weight of these organs. In contrast, potato has heavy storage organs that are supported by the soil. In this mini-review, we introduce strategies of intensifying potato productivity and discuss recent advances in this research area.

Fabrication and optimization of extended-release beads of diclofenac sodium based on Ca++ cross-linked Taro (Colocasia esculenta) stolon polysaccharide and pectin by quality-by-design approach.

The present investigation was aimed to fabricate and optimize extended-release beads of diclofenac sodium based on an ion-cross-linked matrix of pectin (PTN) and taro (Colocasia esculenta) stolon polysaccharide (TSP) with 23 full factorial design. Total polysaccharide concentration (TPC), polysaccharide ratio (PR), and cross-linker concentration ([CaCl2]) were taken as independent factors with two levels of each. Initially, TSP was extracted, purified, and characterized. Fourier-transform infrared spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC) showed drug-polymer compatibility. The study also revealed the significant positive effect of TSP on drug entrapment efficiency (DEE) and sustaining drug release. The response variables (DEE, cumulative % drug-release at 1, 2, 4, 6, and 10 h, release-constant, time for 50 % and 90 % drug release (T50%, T90%), release-similarity factor (f2), and difference factor (f1) were analyzed, and subsequently, independent fabrication variables were numerically optimized by Design-Expert software (Version-13; Stat-Ease Inc., Minneapolis). The optimized batch exhibited appreciable DEE of 88.5 % (± 2.2) and an extended-release profile with significantly higher T50%, T90%, and release-similarity factor (f2) of 4.7 h, 11.4 h, and 71.6, respectively. Therefore, the study exhibited successful incorporation of the novel TSP as a potential alternative adjunct polysaccharide in the pectin-based ion-cross-linked inter-penetrating polymeric network for extended drug release.

Reallocation of Soluble Sugars and IAA Regulation in Association with Enhanced Stolon Growth by Elevated CO2 in Creeping Bentgrass.

Extensive stolon development and growth are superior traits for rapid establishment as well as post-stress regeneration in stoloniferous grass species. Despite the importance of those stoloniferous traits, the regulation mechanisms of stolon growth and development are largely unknown. The objectives of this research were to elucidate the effects of the reallocation of soluble sugars for energy reserves and endogenous hormone levels for cell differentiation and regeneration in regulating stolon growth of a perennial turfgrass species, creeping bentgrass (Agrostis stolonifera L.). Plants were grown in growth chambers with two CO2 concentrations: ambient CO2 concentration (400 ± 10 µmol mol-1) and elevated CO2 concentration (800 ± 10 µmol mol-1). Elevated CO2 enhanced stolon growth through increasing stolon internode number and internode length in creeping bentgrass, as manifested by the longer total stolon length and greater shoot biomass. The content of glucose, sucrose, and fructose as well as endogenous IAA were accumulated in stolon nodes and internodes but not in leaves or roots under elevated CO2 concentration. These results illustrated that the production and reallocation of soluble sugars to stolons as well as the increased level of IAA in stolon nodes and internodes could contribute to the enhancement of stolon growth under elevated CO2 in creeping bentgrass.

Association mapping reveals novel genomic regions controlling some root and stolon traits in tetraploid potato (Solanum tuberosum L.).

Tuber crops have measurable biological variation in root and stolon phenotyping and thus may be utilized to identify genomic regions associated with these variations. This is the first comprehensive association mapping study related to potato root and stolon traits. A diverse panel of 192 tetraploid potato (Solanum tuberosum L.) genotypes were grown in aeroponics to reveal a biologically significant variation and detection of genomic regions associated with the root and stolon traits. Phenotyping of root traits was performed by image analysis software "WinRHIZO" (a root scanning method), and stolon traits was measured manually, while SolCAP 25K potato array was used for genotyping. Significant variation was observed between the potato genotypes for root and stolon traits along with high heritabilities (0.80 in TNS to 0.95 in SL). For marker-trait associations, Q + K linear mixed model was implemented and 50 novel genomic regions were detected. Significantly associated SNPs with stolon traits were located on chr 4, chr 6, chr 7, chr 9, chr 11 and chr 12, while those linked to root traits on chr 1, chr 2, chr 3, chr 9, chr 11, and chr 12. Structure and PCA analysis grouped genotypes into four sub-populations disclosing population genetic diversity. LD decay was observed at 2.316 Mbps (r 2 = 0.29) in the population. The identified SNPs were associated with genes performing vital functions such as root signaling and signal transduction in stress environments (GT-2 factors, protein kinases SAPK2-like and protein phosphatases "StPP1"), transcriptional and post-transcriptional gene regulation (RNA-binding proteins), sucrose synthesis and transporter families (UGPase, Sus3, SuSy, and StSUT1) and PVY resistance (Ry sto). The findings of our study can be employed in future breeding programs for improvement in potato production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02727-6.

Proteome analysis of bermudagrass stolons and rhizomes provides new insights into the adaptation of plant stems to aboveground and underground growth.

As an important perennial warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) forms underground-growing rhizomes and aboveground-growing stolons simultaneously, making it a fast propagating clonal plant with strong regeneration ability. In the current study, we compared the internode proteomes of rhizomes and stolons at the same developmental stage in the bermudagrass cultivar Yangjiang using iTRAQ. The results indicated that 228 protein species were differentially accumulated in the two specialized stems. In agreement with the different contents of starch, chlorophyll, anthocyanin and H2O2 in the two types of stems, photosynthesis and flavonoid biosynthesis were enriched with differentially accumulated protein species (DAPs) in stolons, whereas starch and sucrose metabolism, glycolysis, and H2O2 metabolism were enriched with DAPs in rhizomes. Burying stolons in the soil resulted in the gradual degradation of chlorophyll and anthocyanin, accumulation of starch, and increment of H2O2, which is similar to the physiological characteristics of rhizomes. These results collectively revealed that stolons and rhizomes of bermudagrass have significant differences at the proteome level and light might play important regulatory roles in the discrepancy of the proteome profiles and specialization of the two stems, providing new insights into the adaptation of plant stems to aboveground and underground growth. BIOLOGICAL SIGNIFICANCE: As two types of specialized stems that grow underground and aboveground respectively, rhizomes and stolons play important roles in overwintering and ecological invasion of many perennial and clonal plants. However, because rhizomes and stolons rarely coexist in single plant species, the differences between the two stems remain unclear at the molecular level. In this study, through an iTRAQ comparative proteomic analysis, we reported the identification of 228 differentially accumulated protein species (DAPs) in rhizomes and stolons of bermudagrass for the first time. We found that the 228 DAPs were interconnected to form protein networks in regulating diverse cellular activities and biochemical reactions. We also observed that stolons growing underground showed similar physiological activities and DAP expression as those of underground-growing rhizomes, suggesting that light might play important regulatory roles in the specialization of stolons and rhizomes. These results expanded our understanding of the mysterious adaption of plant stems to different growth conditions.

Organ-Specific Transcriptome Analysis Identifies Candidate Genes Involved in the Stem Specialization of Bermudagrass (Cynodon dactylon L.).

As an important warm-season turfgrass and forage grass species with wide applications, bermudagrass (Cynodon dactylon L.) simultaneously has shoot, stolon and rhizome, three types of stems with different physiological functions. To better understand how the three types of stems differentiate and specialize, we generated an organ-specific transcriptome dataset of bermudagrass encompassing 114,169 unigenes, among which 100,878 and 65,901 could be assigned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Gene Ontology (GO) terms, respectively. Using the dataset, we comprehensively analyzed the gene expression of different organs, especially the shoot, stolon and rhizome. The results indicated that six organs of bermudagrass all contained more than 52,000 significantly expressed unigenes, however, only 3,028 unigenes were enrich-expressed in different organs. Paired comparison analyses further indicated that 11,762 unigenes were differentially expressed in the three types of stems. Gene enrichment analysis revealed that 39 KEGG pathways were enriched with the differentially expressed unigenes (DEGs). Specifically, 401 DEGs were involved in plant hormone signal transduction, whereas 1,978 DEGs were transcription factors involved in gene expression regulation. Furthermore, in agreement with the starch content and starch synthase assay results, DEGs encoding starch synthesis-related enzymes all showed the highest expression level in the rhizome. These results not only provided new insights into the specialization of stems in bermudagrass but also made solid foundation for future gene functional studies in this important grass species and other stoloniferous/rhizomatous plants.

Integrative anatomical study of the branched annelid Ramisyllis multicaudata (Annelida, Syllidae).

The sponge-dwelling Syllidae Ramisyllis multicaudata and Syllis ramosa are the only annelid species for which a branched body with one head and multiple posterior ends is known. In these species, the head is located deep within the sponge, and the branches extend through the canal system of their host. The morphology of these creatures has captivated annelid biologists since they were first discovered in the late XIXth century, and their external characteristics have been well documented. However, how their branched bodies fit within their symbiotic host sponges and how branches translate into internal anatomy has not been documented before. These features are crucially relevant for understanding the body of these animals, and therefore, the aim of this study was to investigate these aspects. In order to assess these questions, live observation, as wells as histology, immunohistochemistry, micro-computed tomography, and transmission electron microscopy techniques were used on specimens of R. multicaudata. By using these techniques, we show that the complex body of R. multicaudata specimens extends greatly through the canal system of their host sponges. We demonstrate that iterative external bifurcation of the body is accompanied by the bifurcation of the longitudinal organ systems that are characteristic of annelids. Additionally, we also highlight that the bifurcation process leaves an unmistakable fingerprint in the form of newly-described "muscle bridges." These structures theoretically allow one to distinguish original and derived branches at each bifurcation. Last, we characterize some of the internal anatomical features of the stolons (reproductive units) of R. multicaudata, particularly their nervous system. Here, we provide the first study of the internal anatomy of a branched annelid. This information is not only crucial to deepen our understanding of these animals and their biology, but it will also be key to inform future studies that try to explain how this morphology evolved.

Spatial Distribution of Medusa Cunina octonaria and Frequency of Parasitic Association with Liriope tetraphylla (Cnidaria: Hydrozoa: Trachylina) in Temperate Southwestern Atlantic Waters.

This study examined the spatial distribution of the medusae phase of Cunina octonaria (Narcomedusae) in temperate Southwestern Atlantic waters using a total of 3,288 zooplankton lots collected along the Uruguayan and Argentine waters (34-56°S), which were placed in the Medusae collection of the Universidad Nacional de Mar del Plata, Argentina. In addition, we reported the peculiar parasitic association between two hydrozoan species: the polypoid phase (stolon and medusoid buds) of C. octonaria (parasite) and the free-swimming medusa of Liriope tetraphylla (Limnomedusae) (host) over a one-year sampling period (February 2014 to March 2015) in the coasts of Mar del Plata, Argentina. We examined the seasonality, prevalence, and intensity of parasitic infection. Metadata associated with the medusa collection was also used to map areas of seasonality where such association was observed. Cunina octonaria was found from southern Uruguay to the coast of Mar del Plata (34.8-38.2°S, 57.2-54.0°W), with the highest abundances and frequency of occurrence in the Río de la Plata estuary. The parasitic association was identified from the austral warm period (spring-summer season) until mid-autumn. Out of the 21,734 L. tetraphylla specimens that were examined, 316 were parasitized (prevalence = 1.5%) exclusively in the manubrium and gastric peduncle, with an infection intensity of 1 to 2 stolons per host. Furthermore, the medusoid buds per stolon ranged from 11 and 29 at different stages of development. No significant differences were observed between the umbrella diameter of parasitized and non-parasitized L. tetraphylla specimens, nor was any significant correlation identified between umbrella diameter and prevalence, and intensity of infection. According to the aggregation coefficient, C. octonaria had an overdispersed distribution in the host population. All parasitized hosts showed stomach vacuity due to the location of the stolon, which blocked the mouth of the host. We identified the parasitic association in the coasts of Mar del Plata, as well as in both coasts of the Río de la Plata Estuary (Uruguayan-Argentinean coasts). In the Southwestern Atlantic, several biological interactions between medusae and other groups have been identified; however, the specific host selectivity of C. octonaria for L. tetraphylla was not previously identified. Here we discuss the ecological importance of this association during the holoplanktonic life history of the narcomedusae. Additionally, we report the southern limit of the spatial distribution of this particular parasitic association in the Southwestern Atlantic, thus increasing the knowledge of biological associations of gelatinous zooplankton (Cnidaria and Ctenophora) on Uruguayan and Argentinean coasts.

Filamentous Connections between Ediacaran Fronds.

Fossils of the Ediacaran macrobiota (∼571-539 mya) record phylogenetically diverse marine palaeocommunities, including early animals, which pre-date the "Cambrian Explosion" [1-4]. Benthic forms with a frondose gross morphology, assigned to the morphogroups Rangeomorpha [5] and Frondomorpha (see also Arboreomorpha) [6-8], are among the most temporally wide-ranging and environmentally tolerant members of the Ediacaran macrobiota [6] and dominated deep-marine ecosystems ∼571-560 mya [9-11]. Investigations into the morphology [12-14], palaeoecology [10, 15, 16], reproductive strategies [17, 18], feeding methods [9, 19], and morphogenesis of frondose taxa together constrain their phylogenetic position to the metazoan (for Rangeomorpha) or eumetazoan (e.g., Arborea) total groups [14, 20], but tighter constraint is currently lacking. Here, we describe fossils of abundant filamentous organic structures preserved among frond-dominated fossil assemblages in Newfoundland (Canada). The filaments constitute a prominent component of the ecosystems, and exhibit clear physical associations with at least seven frondose taxa. Individual specimens of one uniterminal rangeomorph taxon appear to be directly connected by filaments across distances of centimeters to meters. Such physical linkages are interpreted to reflect evidence for stolonic connections: a conclusion with potential implications for the phylogenetic placement and palaeoecology of frondose organisms. Consideration of extant stoloniferous organisms suggests that Ediacaran frondose taxa were likely clonal and resurrects the possibility that they may have been colonial (e.g., [21, 22]). VIDEO ABSTRACT.