Molecular, cellular, and developmental foundations of grass diversity.

Humans have cultivated grasses for food, feed, beverages, and construction materials for millennia. Grasses also dominate the landscape in vast parts of the world, where they have adapted morphologically and physiologically, diversifying to form ~12,000 species. Sequences of hundreds of grass genomes show that they are essentially collinear; nonetheless, not all species have the same complement of genes. Here, we focus on the molecular, cellular, and developmental bases of grain yield and dispersal-traits that are essential for domestication. Distinct genes, networks, and pathways were selected in different crop species, reflecting underlying genomic diversity. With increasing genomic resources becoming available in nondomesticated species, we anticipate advances in coming years that illuminate the ecological and economic success of the grasses.

On the Origin of Tetraploid Vernal Grasses (Anthoxanthum) in Europe.

Polyploidy has played a crucial role in the evolution of many plant taxa, namely in higher latitudinal zones. Surprisingly, after several decades of an intensive research on polyploids, there are still common polyploid species whose evolutionary history is virtually unknown. Here, we addressed the origin of sweet vernal grass (Anthoxanthum odoratum) using flow cytometry, DNA sequencing, and in situ hybridization-based cytogenetic techniques. An allotetraploid and polytopic origin of the species has been verified. The chromosome study reveals an extensive variation between the European populations. In contrast, an autopolyploid origin of the rarer tetraploid vernal grass species, A. alpinum, has been corroborated. Diploid A. alpinum played an essential role in the polyploidization of both European tetraploids studied.

Water vapor sorption behavior of bamboo pertaining to its hierarchical structure.

Bamboo is an anisotropic, hierarchical, and hygroscopic material. Moisture transport in bamboo is one of the most fundamental properties affecting almost all other physical and mechanical properties of the material. This study investigated the water vapor sorption behaviors of bamboo at various structural levels: cell walls, cells (with pits) and bamboo blocks. The specimens with two sorption directions, longitudinal (L) and transverse (T), were measured by saturated salt solution method and dynamic vapor sorption. The parallel exponential kinetics model was used to analyze the sorption kinetics. The results showed that at the cell wall level, the sorption rate and equilibrium moisture content (EMC) of cell wall in the L specimens were larger than those in the T specimens. The differences were probably caused by the looser cell wall layers in the L specimens. At the cellular scale, pits in the cell wall resulted in an enhanced sorption rate and EMC of the T specimens compared with the L specimens where the pits in the parenchyma cells were only distributed in the lateral walls but not in end walls. At the macro scale, the sorption rate and moisture content of bamboo blocks were largely controlled by the vessel cells. As a hierarchically-structured plant, bamboo performs the biological function of moisture transport at all these scales. This work helps improve the understanding of water transport behavior in bamboo, which may lead to better bamboo drying and impregnation processes.

Bioproduction of 4-Vinylphenol and 4-Vinylguaiacol ß-Primeverosides Using Transformed Bamboo Cells Expressing Bacterial Phenolic Acid Decarboxylase.

Phenolic acid decarboxylase (PAD) catalyzes the decarboxylation of hydroxycinnamic acids to produce hydroxystyrenes, which serve as starting materials for the production of polymers. Bamboo (Phyllostachys nigra; Pn) cells, a suitable host for producing phenylpropanoid-derived compounds, were transformed to express PAD of Bacillus amyloliquefaciens (BaPAD). BaPAD-transformed cells accumulated several metabolites that were not detected in wild-type Pn cells or BaPAD-negative transformant. Two major metabolites were isolated from BaPAD-transformed cells, and elucidation of their chemical structures confirmed these as 4-vinylphenol ß-primeveroside (4-VPP) and 4-vinylguaiacol ß-primeveroside (4-VGP). The production titers of 4-VPP and 4-VGP reached 48 and 33 mg/L at the maximum, respectively. Feeding experiments with 4-vinylphenol (4-VP), 4-vinylguaiacol (4-VG), and their glucosides indicated that 4-VPP and 4-VGP are formed by sequential glycosylation of 4-VP and 4-VG via their corresponding glucosides. Our results demonstrate the versatility of Pn cells for producing styrene derivatives, and indicate the presence of a unique glycosylation pathway to produce 4-VPP and 4-VGP in Pn cells.

Soil PAHs contamination effect on the cellular and subcellular organelle changes of Phragmites australis Cav.

The concentrations of ∑16 priority polycyclic aromatic hydrocarbons (PAHs) for soils, roots, and above-ground parts of reed (Phragmites australis Cav.) were determined on different monitoring plots located near the city of Kamensk-Shakhtinsky, southern Russia, where historically received industrial sewage and sludge. The total PAHs concentration in monitoring soil plots was significantly higher than those in the background site which situated at the distance of 2 km from the contamination source. Accordingly, the maximum accumulation was found for phenanthrene and chrysene among the 16 priority PAHs in most of the plant samples collected in the impact zone. The effects of PAHs' pollution on changes of Phragmites australis Cav. cellular and subcellular organelles in the studied monitoring sites were also determined using optical and electron microscopy, respectively. The obtained data showed that increasing of PAHs contamination negatively affected the ultrastructural changes of the studied plants. Phragmites australis Cav. showed a high level of adaptation to the effect of stressors by using tissue and cell levels. In general, the detected alterations under the PAHs effect were possibly connected to changes in biochemical and histochemical parameters as a response for reactive oxygen species and as a protective response against oxidative stress. The obtained results introduce innovative findings of cellular and subcellular changes in plants exposed to ∑16 priority PAHs as very persistent and toxic contaminants.

An image processing method for investigating the morphology of cereal endosperm cells.

In cereal seeds, the number, morphology and development of endosperm cells are closely related to grain quality, weight and yield. Endosperm cells differ morphologically in different regions of the seed. Nevertheless, it is important to be able to analyze the morphology of cereal endosperm cells. We established an image processing method to enhance the outlines of endosperm cells. The endosperm cell wall was traced precisely using the "pen tool" in Photoshop software (PS). The tracing was defined as the "work path" and was highlighted using the PS "brush tool." Images of mature rice, maize and wheat endosperm sections stained with different methods were analyzed using this method. Combined with the whole sections of mature and developing cereal kernels, the processed image exhibited clearly the morphology of endosperm cells in any region of endosperm and at any stage of endosperm development. The processed image was more accurate and efficient for analyzing morphological characteristics than the unprocessed image.

Form, development and function of grass stomata.

Stomata are cellular breathing pores on leaves that open and close to absorb photosynthetic carbon dioxide and to restrict water loss through transpiration, respectively. Grasses (Poaceae) form morphologically innovative stomata, which consist of two dumbbell-shaped guard cells flanked by two lateral subsidiary cells (SCs). This 'graminoid' morphology is associated with faster stomatal movements leading to more water-efficient gas exchange in changing environments. Here, we offer a genetic and mechanistic perspective on the unique graminoid form of grass stomata and the developmental innovations during stomatal cell lineage initiation, recruitment of SCs and stomatal morphogenesis. Furthermore, the functional consequences of the four-celled, graminoid stomatal morphology are summarized. We compile the identified players relevant for stomatal opening and closing in grasses, and discuss possible mechanisms leading to cell-type-specific regulation of osmotic potential and turgor. In conclusion, we propose that the investigation of functionally superior grass stomata might reveal routes to improve water-stress resilience of agriculturally relevant plants in a changing climate.

Cellular and molecular characterization of a thick-walled variant reveal a pivotal role of shoot apical meristem in transverse development of bamboo culm.

Little is known about the mechanisms underlying the development of bamboo culm. Using anatomical, mathematical modeling, and genomics methods, we investigated the role of shoot apical meristem (SAM) in the development of the transverse morphology of bamboo culm and explored the underlying cellular and molecular processes. We discovered that maintenance of SAM morphology that can produce circular culm and increase in SAM cell numbers, especially corpus cells, is the means by which bamboo makes a larger culm with a regular pith cavity and culm wall during development. A less cellular form of SAM with a lower proportion of corpus cells causes an abnormal higher ratio of wall component cells to pith cells, which breaks the balance of their interaction and triggers the random invasion of wall component cells into pith tissues during development, and finally results in the various thick culm walls of Phyllostachys nidularia f. farcta. The smaller SAM also results in a lower level of hormones such as cytokinin and auxin, and down-regulates hormone signaling and the downstream functional genes such as those related to metabolism, which finally results in a dwarf and smaller diameter culm with lower biomass. These results provide an important perspective on the culm development of bamboo, and support a plausible mechanism causing the size-reduced culm and various thick culm walls of P. nidularia f. farcta.

The highly divergent Jekyll genes, required for sexual reproduction, are lineage specific for the related grass tribes Triticeae and Bromeae.

Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific.

Rational metabolic-flow switching for the production of exogenous secondary metabolites in bamboo suspension cells.

The synthetic biology-driven production of high-value plant secondary metabolites in microbial hosts has attracted extensive attention despite various challenges, including correct protein expression and limited supplies of starting materials. In contrast, plant cell cultures are rarely used for this purpose owing to their slow proliferation rates and laborious transformation processes. Here, we propose a "rational metabolic-flow switching" strategy to efficiently produce exogenous secondary metabolites using suspension-cultured bamboo (Phyllostachys nigra; Pn) cells as model production hosts. The Pn cells biosynthesise hydroxycinnamic acid amides (HCAAs) of putrescine as major secondary metabolites, which indicates that the phenylpropanoid and polyamine biosynthetic pathways are highly active and that the Pn cells may produce alternative secondary metabolites derived from those pathways. Stable transformants of Pn cells expressing agmatine coumaroyltransferase of barley (Hordeum vulgare) were generated with the expectation of metabolic-flow switching from HCAAs of putrescine to those of agmatine. In the recombinant Pn cells, the levels of HCAAs of putrescine decreased and the HCAAs of agmatine were produced instead. The production titre of the major product, p-coumaroylagmatine, reached approximately 360 mg/L, providing a proof-of-concept for the usefulness of "rational metabolic-flow switching" in synthetic biology using plant cell hosts.

Transcriptional alterations during proliferation and lignification in Phyllostachys nigra cells.

Highly-lignified culms of bamboo show distinctive anatomical and mechanical properties compared with the culms of other grass species. A cell culture system for Phyllostachys nigra has enabled investigating the alterations in cellular states associated with secondary cell wall formation during its proliferation and lignification in woody bamboos. To reveal transcriptional changes related to lignification in bamboo, we analyzed transcriptome in P. nigra cells treated with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the synthetic cytokinin benzylaminopurine (BA) by RNA-seq analysis. We found that some genes putatively involved in cell wall biogenesis and cell division were up-regulated in response to the 2,4-D treatment, and the induction of lignification by the BA treatment was correlated with up-regulation of genes involved in the shikimate pathway. We also found that genes encoding MYB transcription factors (TFs) show correlated expression patterns with those encoding cinnamyl alcohol dehydrogenase (CAD), suggesting that MYB TFs presumably regulate secondary cell wall formation in the bamboo cells. These findings suggest that cytokinin signaling may regulate lignification in P. nigra cells through coordinated transcriptional regulation and metabolic alterations. Our results have also produced a useful resource for better understanding of secondary cell wall formation in bamboo plants.

Comparative Analyses of Anatomical Structure, Phytohormone Levels, and Gene Expression Profiles Reveal Potential Dwarfing Mechanisms in Shengyin Bamboo (Phyllostachys edulis f. tubaeformis).

Moso bamboo (Phyllostachys edulis) is one of the most important bamboo species in China and the third most important plant species for timber production. However, the dwarf variant of moso bamboo, P. edulis f. tubaeformis (shengyin bamboo), which has shortened internodes, is not well studied. We used anatomical, hormonal, and transcriptomic approaches to study internode shortening and shoot growth in dwarf shengyin and wild moso bamboo. Phenotypic and anatomical observations showed that dwarfing in shengyin bamboo is due to reduced internode length, and the culm fibers in shengyin bamboo are significantly shorter and thicker than in wild moso bamboo. We measured the levels of endogenous hormones in the internodes and found that shengyin bamboo had lower levels of four hormones while two others were higher in wild moso bamboo. Comparative transcriptome analyses revealed a potential regulating mechanism for internode length involving genes for cell wall loosening-related enzymes and the cellulose and lignin biosynthesis pathways. Genes involved in hormone biosynthesis and signal transduction, especially those that showed significant differential expression in the internodes between shengyin and wild moso bamboo, may be important in determining the shortened internode phenotype. A hypothesis involving possible cross-talk between phytohormone signaling cues and cell wall expansion leading to dwarfism in shengyin bamboo is proposed. The results presented here provide a comprehensive exploration of the biological mechanisms that determine internode shortening in moso bamboo.

Tapetal-Delayed Programmed Cell Death (PCD) and Oxidative Stress-Induced Male Sterility of Aegilops uniaristata Cytoplasm in Wheat.

Cytoplasmic male sterility (CMS) plays a crucial role in the utilization of hybrid vigor. Pollen development is often accompanied by oxidative metabolism responses and tapetal programmed cell death (PCD), and deficiency in these processes could lead to male sterility. Aegilops uniaristata cytoplasmic male sterility (Mu-CMS) wheat is a novel male-sterile line in wheat, which possess important potential in hybrid wheat breeding. However, its CMS mechanisms remain poorly understood. In our study, U87B1-706A, with the Aegilops uniaristata cytoplasm, and the maintainer line 706B were used to explore the abortive reason. Compared with 706B, histological analysis and PCD detection of the anther demonstrated that U87B1-706A appeared as delayed tapetal PCD as well as a disorganized organelle phenotype in the early uninucleate stage. Subsequently, a shrunken microspore and disordered exine structure were exhibited in the late uninucleate stage. While the activities of antioxidase increased markedly, the nonenzymatic antioxidant contents declined obviously following overacummulation of reactive oxygen species (ROS) during pollen development in U87B1-706A. Real-time quantitative PCR testified that the transcript levels of the superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) genes, encoding pivotal antioxidant enzymes, were up-regulated in early pollen development. Therefore, we deduce excess ROS as a signal may be related to the increased expression levels of enzyme genes, thereby breaking the antioxidative system balance, resulting in delayed tapetal PCD initiation, which finally led to pollen abortion and male sterility in U87B1-706A. These results provide evidence to further explore the mechanisms of abortive pollen in CMS wheat.

Distinct wall polymer deconstruction for high biomass digestibility under chemical pretreatment in Miscanthus and rice.

Miscanthus is a leading bioenergy crop and rice provides enormous biomass for biofuels. Using Calcofluor White staining, this work in situ observed an initial lignocellulose hydrolysis in two distinct Miscanthus accessions, rice cultivar (NPB), and Osfc16 mutant after mild chemical pretreatments. In comparison, the M. sin and Osfc16 respectively exhibited weak Calcofluor fluorescence compared to the M. sac and NPB during enzymatic hydrolysis, consistent with the high biomass saccharification detected in vitro. Using xyloglucan-directed monoclonal antibodies (mAbs), xyloglucan deconstruction was observed from initial cellulose hydrolysis, whereas the M. sin and Osfc16 exhibited relatively strong immunolabeling using xylan-directed mAb, confirming previous findings of xylan positive impacts on biomass saccharification. Furthermore, the M. sin showed quick disappearance of RG-I immunolabeling with varied HG labelings between acid and alkali pretreatments. Hence, this study demonstrated a quick approach to explore wall polymer distinct deconstruction for enhanced biomass saccharification under chemical pretreatment in bioenergy crops.

Stomatal development: focusing on the grasses.

The development and patterning of stomata in the plant epidermis has emerged as an ideal system for studying fundamental plant developmental processes. Over the past twenty years most studies of stomata have used the model dicotyledonous plant Arabidopsis thaliana. However, cultivated monocotyledonous grass (or Gramineae) varieties provide the majority of human nutrition, and future research into grass stomata could be of critical importance for improving food security. Recent studies using Brachypodium distachyon, Hordeum vulgare (barley) and Oryza sativa (rice) have led to the identification of the core transcriptional regulators essential for stomatal initiation and progression in grasses, and begun to unravel the role of secretory signaling peptides in controlling stomatal developmental. This review revisits how stomatal developmental unfolds in grasses, and identifies key ontogenetic steps for which knowledge of the underpinning molecular mechanisms remains outstanding.

Branched Pectic Galactan in Phloem-Sieve-Element Cell Walls: Implications for Cell Mechanics.

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a ß-1,6-galactosyl substitution of ß-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear ß-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.

Genome-wide analysis and transcriptomic profiling of the auxin biosynthesis, transport and signaling family genes in moso bamboo (Phyllostachys heterocycla).

BACKGROUND: Auxin is essential for plant growth and development. Although substantial progress has been made in understanding auxin pathways in model plants such as Arabidopsis and rice, little is known in moso bamboo which is famous for its fast growth resulting from the rapid cell elongation and division. RESULTS: Here we showed that exogenous auxin has strong effects on crown and primary roots. Genes involved in auxin action, including 13 YUCCA (YUC) genes involved in auxin synthesis, 14 PIN-FORMED/PIN-like (PIN/PILS) and 7 AUXIN1/LIKE-AUX1 (AUX1/LAX) members involved in auxin transport, 10 auxin receptors (AFB) involved in auxin perception, 43 auxin/indole-3-aceticacid (AUX/IAA) genes, and 41 auxin response factors (ARF) involved in auxin signaling were identified through genome-wide analysis. Phylogenetic analysis of these genes from Arabidopsis, Oryza sativa and bamboo revealed that auxin biosynthesis, transport, and signaling pathways are conserved in these species. A comprehensive study of auxin-responsive genes using RNA sequencing technology was performed, and the results also supported that moso bamboo shared a conserved regulatory mechanism for the expression of auxin pathway genes; meanwhile it harbors its own specific properties. CONCLUSIONS: In summary, we generated an overview of the auxin pathway in bamboo, which provides information for uncovering the precise roles of auxin pathway in this important species in the future.

Evolutionary modes of emergence of short interspersed nuclear element (SINE) families in grasses.

Short interspersed nuclear elements (SINEs) are non-autonomous transposable elements which are propagated by retrotransposition and constitute an inherent part of the genome of most eukaryotic species. Knowledge of heterogeneous and highly abundant SINEs is crucial for de novo (or improvement of) annotation of whole genome sequences. We scanned Poaceae genome sequences of six important cereals (Oryza sativa, Triticum aestivum, Hordeum vulgare, Panicum virgatum, Sorghum bicolor, Zea mays) and Brachypodium distachyon to examine the diversity and evolution of SINE populations. We comparatively analyzed the structural features, distribution, evolutionary relation and abundance of 32 SINE families and subfamilies within grasses, comprising 11 052 individual copies. The investigation of activity profiles within the Poaceae provides insights into their species-specific diversification and amplification. We found that Poaceae SINEs (PoaS) fall into two length categories: simple SINEs of up to 180 bp and dimeric SINEs larger than 240 bp. Detailed analysis at the nucleotide level revealed that multimerization of related and unrelated SINE copies is an important evolutionary mechanism of SINE formation. We conclude that PoaS families diversify by massive reshuffling between SINE families, likely caused by insertion of truncated copies, and provide a model for this evolutionary scenario. Twenty-eight of 32 PoaS families and subfamilies show significant conservation, in particular either in the 5' or 3' regions, across Poaceae species and share large sequence stretches with one or more other PoaS families.

Genetic complexity of miscanthus cell wall composition and biomass quality for biofuels.

BACKGROUND: Miscanthus sinensis is a high yielding perennial grass species with great potential as a bioenergy feedstock. One of the challenges that currently impedes commercial cellulosic biofuel production is the technical difficulty to efficiently convert lignocellulosic biomass into biofuel. The development of feedstocks with better biomass quality will improve conversion efficiency and the sustainability of the value-chain. Progress in the genetic improvement of biomass quality may be substantially expedited by the development of genetic markers associated to quality traits, which can be used in a marker-assisted selection program. RESULTS: To this end, a mapping population was developed by crossing two parents of contrasting cell wall composition. The performance of 182 F1 offspring individuals along with the parents was evaluated in a field trial with a randomized block design with three replicates. Plants were phenotyped for cell wall composition and conversion efficiency characters in the second and third growth season after establishment. A new SNP-based genetic map for M. sinensis was built using a genotyping-by-sequencing (GBS) approach, which resulted in 464 short-sequence uniparental markers that formed 16 linkage groups in the male map and 17 linkage groups in the female map. A total of 86 QTLs for a variety of biomass quality characteristics were identified, 20 of which were detected in both growth seasons. Twenty QTLs were directly associated to different conversion efficiency characters. Marker sequences were aligned to the sorghum reference genome to facilitate cross-species comparisons. Analyses revealed that for some traits previously identified QTLs in sorghum occurred in homologous regions on the same chromosome. CONCLUSION: In this work we report for the first time the genetic mapping of cell wall composition and bioconversion traits in the bioenergy crop miscanthus. These results are a first step towards the development of marker-assisted selection programs in miscanthus to improve biomass quality and facilitate its use as feedstock for biofuel production.

Root hair development in grasses and cereals (Poaceae).

Root hairs are tubular, cellular outgrowths of epidermal cells that extend from the root surface into the soil. Root hairs tether root systems to their growth substrate, take up inorganic nutrients and water, and interact with the soil microflora. At maturity, the root epidermis comprises two cell types; cells with root hairs and hairless epidermal cells. These two cell types alternate with each other along longitudinal files in grasses and cereals (Poaceae). While the mechanism by which this alternating pattern develops is unknown, the later stages of root hair differentiation are controlled by a conserved mechanism that promotes root hair development among angiosperms.