Dehiscence Zone Research Articles

A Model for the Gene Regulatory Network Along the Arabidopsis Fruit Medio-Lateral Axis: Rewiring the Pod Shatter Process.

Different convergent evolutionary strategies adopted by angiosperm fruits lead to diverse functional seed dispersal units. Dry dehiscent fruits are a common type of fruit, characterized by their lack of fleshy pericarp and the release of seeds at maturity through openings (dehiscence zones, DZs) in their structure. In previous decades, a set of core players in DZ formation have been intensively characterized in Arabidopsis and integrated in a gene regulatory network (GRN) that explains the morphogenesis of these tissues. In this work, we compile all the experimental data available to date to build a discrete Boolean model as a mechanistic approach to validate the network and, if needed, to identify missing components of the GRN and/or propose new hypothetical regulatory interactions, but also to provide a new formal framework to feed further work in Brassicaceae fruit development and the evolution of seed dispersal mechanisms. Hence, by means of exhaustive in-silico validations and experimental evidence, we are able to incorporate both the NO TRANSMITTING TRACT (NTT) transcription factor as a new additional node, and a new set of regulatory hypothetical rules to uncover the dynamics of Arabidopsis DZ specification.

Growth and tension in explosive fruit

Exploding seed pods of the common weed Cardamine hirsuta have the remarkable ability to launch seeds far from the plant. The energy for this explosion comes from tension that builds up in the fruit valves. Above a critical threshold, the fruit fractures along its dehiscence zone and the two valves coil explosively, ejecting the seeds. A common mechanism to generate tension is drying, causing tissues to shrink. However, this does not happen in C.hirsuta fruit. Instead, tension is produced by active contraction of growing exocarp cells in the outer layer of the fruit valves. Exactly how growth causes the exocarp tissue to contract and generate pulling force is unknown. Here we show that the reorientation of microtubules in the exocarp cell cortex changes the orientation of cellulose microfibrils in the cell wall and the consequent cellular growth pattern. We used mechanical modeling to show how tension emerges through growth due to the highly anisotropic orientation of load-bearing cellulose microfibrils and their effect on cell shape. By explicitly defining the cell wall as multi-layered in our model, we discovered that a cross-lamellate pattern of cellulose microfibrils further enhances the developing tension in growing cells. Therefore, the interplay of cell wall properties with turgor-driven growth enables the fruit exocarp to generate sufficient tension to power explosive seed dispersal.

Evolution and development of fruits of Erycina pusilla and other orchid species.

Fruits play a crucial role in seed dispersal. They open along dehiscence zones. Fruit dehiscence zone formation has been intensively studied in Arabidopsis thaliana. However, little is known about the mechanisms and genes involved in the formation of fruit dehiscence zones in species outside the Brassicaceae. The dehiscence zone of A. thaliana contains a lignified layer, while dehiscence zone tissues of the emerging orchid model Erycina pusilla include a lipid layer. Here we present an analysis of evolution and development of fruit dehiscence zones in orchids. We performed ancestral state reconstructions across the five orchid subfamilies to study the evolution of selected fruit traits and explored dehiscence zone developmental genes using RNA-seq and qPCR. We found that erect dehiscent fruits with non-lignified dehiscence zones and a short ripening period are ancestral characters in orchids. Lignified dehiscence zones in orchid fruits evolved multiple times from non-lignified zones. Furthermore, we carried out gene expression analysis of tissues from different developmental stages of E. pusilla fruits. We found that fruit dehiscence genes from the MADS-box gene family and other important regulators in E. pusilla differed in their expression pattern from their homologs in A. thaliana. This suggests that the current A. thaliana fruit dehiscence model requires adjustment for orchids. Additionally, we discovered that homologs of A. thaliana genes involved in the development of carpel, gynoecium and ovules, and genes involved in lipid biosynthesis were expressed in the fruit valves of E. pusilla, implying that these genes may play a novel role in formation of dehiscence zone tissues in orchids. Future functional analysis of developmental regulators, lipid identification and quantification can shed more light on lipid-layer based dehiscence of orchid fruits.

Transcriptomic Time-Course Sequencing: Insights into the Cell Wall Macromolecule-Mediated Fruit Dehiscence during Ripening in Camellia oleifera.

Camellia oleifera (C. oleifera), one of the world's four major edible woody oil crops, has been widely planted in southern China's subtropical region for the extremely high nutritional and health benefits of its seed oil. Timing and synchronization of fruit dehiscence are critical factors influencing the oil output and quality, as well as the efficiency and cost of harvesting C. oleifera, yet they extremely lack attention. To gain an understanding of the molecular basis underlying the dehiscence of C. oleifera fruit, we sampled pericarp-replum tissues containing dehiscence zones from fruits at different developmental stages and performed time-series transcriptomic sequencing and analysis for the first time. Statistical and GO enrichment analysis of differentially expressed genes revealed that drastic transcriptional changes occurred over the last short sampling interval (4 days, 18th-22nd October), which directed functional classifications link to cell wall and cell wall macromolecule activity. WGCNA further showed that factors controlling cell wall modification, including endo-1,3;1,4-beta-D-glucanase, WAT1-like protein 37, LRR receptor-like serine/threonine-protein kinase, and cellulose synthase A catalytic subunit, were identified as core members of the co-expression network of the last stage highly related modules. Furthermore, in these modules, we also noted genes that were annotated as coding for polygalacturonase and pectinesterase, two pectinases that were expected to be major players in cell separation during dehiscence. qRT-PCR further confirmed the expression profiles of these cell wall modification relating factors, which possessed a special high transcriptional abundance at the final stage. These results suggested the cell wall associated cell separation, one of the essential processes downstream of fruit dehiscence, happened in dehiscing fruit of C. oleifera during ripening. Hydrolases acting on cell wall components are good candidates for signal mediating dehiscence of C. oleifera fruit. In conclusion, our analysis provided insights into the cell wall macromolecule-mediated fruit dehiscence during ripening in C. oleifera.

Difference in Kernel Shape and Endocarp Anatomy Promote Dehiscence in Pistachio Endocarp

A fully split shell in pistachio (Pistacia vera) is a trait that is preferred by consumers and is a criterion in evaluating the grade of the pistachio nut. However, although the expanding kernel has been hypothesized to provide the physical force needed for shell split, the mechanisms that control shell split remain unknown. Furthermore, it is intriguing how the shell, or endocarp, splits at the suture ridge when there is no clear dehiscence zone. The objectives of this study were 1) to identify traits associated with dehiscence in fruit in the high-split rate cultivar Golden Hills when compared with the lower split rate cultivar Kerman and determine the anatomic features associated with endocarp dehiscence at the suture region, and 2) to examine the effect of kernel shape on endocarp dehiscence. We determined that, despite the fact that the pistachio endocarp is composed primarily of a single type of polylobate sclerenchyma cell, specialization of cell shape and size at the suture site results in smaller, more flattened cells. We report there is a furrowing of the shell at the dorsal and apical suture sites, where dehiscence initiates. This furrowing is not observed at the ventral suture site or in the indehiscent fruit of Pistacia atlantica, a species that has been used as rootstock for P. vera. In addition, the size of the kernel in the sagittal axis (the width) is strongly associated with a greater split rate. Based on our results, a tentative model emerges in which, in the absence of specialized cell types, cell shape modification can create an anatomically distinct region that is mechanically weak in the endocarp for the initiation of dehiscence, whereas the force from the width of the kernel is necessary for the shell split rate difference as observed in cultivars.

GENOME-WIDE ANALYSES OF CORE REGULATORY MODULE SHATTERING CASCADE GENES IN CANOLA (BRASSICA NAPUS L.)

Premature seed shattering in canola causes massive losses in yield by up to 50% in adverse climatic conditions. In the model plant Arabidopsis thaliana, which belongs to the same family as canola, the Brassicaceae, eight genes participate in a shattering cascade. Phylogenetic reconstruction, syntenic relationships, genomics loci, promoter sequences, and identification of transcription factor-binding sites (TFBSs) faced shattering cascade genes’ analysis. Among these, three genes, SHATTERPROOF1, SHATTERPROOF2, and FRUITFUL (SHP1, SHP2, FUL), belonged to a MADS-box family implicated in fruit dehiscence zone and valve margin constitute a core regulatory module. But, in Brassica, the exact number of genes involved in shattering remained obscure. Grouping BnSHP1-N, BnSHP2-N, and BnFUL-N into their respective clades was according to phylogenetic reconstruction of core regulatory modules (SHP1, SHP2, and FUL) and from other species homologs. The eight shattering cascade genes showed no conservation, indicating their involvement in crushing through separate pathways. The increased number of homologs/paralogs in Brassica was due to occurrences of genome duplication or a triplication event during evolution. Exonization and intronization could be responsible for a variable number and size of the exons and introns in gene structures. Comparative genome synteny analysis of SHP1, SHP2, and FUL revealed correlation and evolutionary insights into gene region relationships in all Brassicaceae. Study results provided basic information on cloning, phylogenetic reconstruction, genomics loci, and identifying transcription factor-binding sites (TFBSs) of core regulatory module genes that might be helpful for developing shattering-resistant genome-edited plants to prevent future yield losses in canola.

Microscopical Analysis of Autofluorescence as a Complementary and Useful Method to Assess Differences in Anatomy and Structural Distribution Underlying Evolutive Variation in Loss of Seed Dispersal in Common Bean.

The common bean has received attention as a model plant for legume studies, but little information is available about the morphology of its pods and the relation of this morphology to the loss of seed dispersal and/or the pod string, which are key agronomic traits of legume domestication. Dehiscence is related to the pod morphology and anatomy of pod tissues because of the weakening of the dorsal and ventral dehiscence zones and the tensions of the pod walls. These tensions are produced by the differential mechanical properties of lignified and non-lignified tissues and changes in turgor associated with fruit maturation. In this research, we histologically studied the dehiscence zone of the ventral and dorsal sutures of the pod in two contrasting genotypes for the dehiscence and string, by comparing different histochemical methods with autofluorescence. We found that the secondary cell wall modifications of the ventral suture of the pod were clearly different between the dehiscence-susceptible and stringy PHA1037 and the dehiscence-resistant and stringless PHA0595 genotypes. The susceptible genotype had cells of bundle caps arranged in a more easily breakable bowtie knot shape. The resistant genotype had a larger vascular bundle area and larger fibre cap cells (FCCs), and due to their thickness, the external valve margin cells were significantly stronger than those from PHA1037. Our findings suggest that the FCC area, and the cell arrangement in the bundle cap, might be partial structures involved in the pod dehiscence of the common bean. The autofluorescence pattern at the ventral suture allowed us to quickly identify the dehiscent phenotype and gain a better understanding of cell wall tissue modifications that took place along the bean's evolution, which had an impact on crop improvement. We report a simple autofluorescence protocol to reliably identify secondary cell wall organization and its relationship to the dehiscence and string in the common bean.

Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins.

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.

GENETIC AND PHYSIOLOGICAL ASPECTS OF SILIQUE SHATTERING IN RAPESEED AND MUSTARD

Rapeseed (Brassica napus L.) and mustard (Brassica juncea L.) are two important oilseed crops grown worldwide for edible oil and meal production, as well as, a source of renewable energy. Silique shattering at the maturity stage is the major cause of seed yield reduction in brassica. Losses in seed yield are more in developing countries due to poor management and the non-availability of combine harvesters. Silique shattering resistance is essential for achieving good seed yield especially in Brassica napus. The silique on plants of rapeseed and mustard mature in different phases due to indeterminate growth habit, which is also a reason for shattering losses. Silique shattering is linked with the creation of a dehiscence zone in a brassica pod. When the siliqua wall loses its hydration, along the length of the siliqua, a few cell layers separate the replum from the pericarp tip of the two silique valves. In the dehiscence zone, it involves the collapse of cell walls and cell separation, as well as, the destruction of the middle lamella and enhanced hydrolytic enzyme activity. To avoid seed yield losses, resistance against silique shattering is essential in rapeseed and mustard cultivars. There are multiple QTLs discovered that control variance in silique shattering. Previous studies validated the shattering process in the model plant Arabidopsis thaliana was controlled by eight different genes. However, their role in controlling silique shattering in rapeseed and mustard is unknown. Modern tools of mutation breeding and genetic engineering, especially CRISPR/Cas9 technology, can be utilized to identify the genetic source for shattering resistance in rapeseed and mustard, which will be helpful for the development of silique-shattering

English

Brassica is the second-largest oilseed crop after Soybean. The total production of Brassica in the overall world is 71 million tons. In Pakistan, its total production per unit area is very low. Biotic and abiotic stresses mainly affect the brassica crop. In agriculture, shattering is the dispersal of crops seeds before their ripening. The pod wall shatters and breaks apart when it loses its hydration and cells split in a dehiscence zone organized at a suture between the edge of the lignified pod and the vascular tissue replum. The degeneration of middle lamella and loss of cellular cohesion in the dehiscence zone are the main reasons for pod shattering and seed losses. Grain yield losses in Brassica vary from 10 to 25 percent due to shattering. More than 400 kg has-1 or 12% seed losses can be occurred due to pod shattering under unfavorable conditions. Insect pest and disease damage also accelerate ripening and pod shattering. The main breeding techniques for developing rapeseed grain yield potential are a good knowledge and application of the morphological, physiological, and genetic basis of grain yield. Modern technologies, such as embryo rescue, marker-assisted breeding, and novel variation (mutation), may make it much simpler to introduce new rapeseed types having shattering tolerance than traditional methods. Thus, an overview of anatomical and physiological aspects and genetics of shattering is presented in the context of recent advances in molecular genetics and several agronomic managements to avoid shattering in Brassica.

Pectin Modification in Seed Coat Mucilage by In Vivo Expression of Rhamnogalacturonan-I- and Homogalacturonan-Degrading Enzymes.

The cell wall is essential for plant survival. Determining the relationship between cell wall structure and function using mutant analysis or overexpressing cell wall-modifying enzymes has been challenging due to the complexity of the cell wall and the appearance of secondary, compensatory effects when individual polymers are modified. In addition, viability of the plants can be severely impacted by wall modification. A useful model system for studying structure-function relationships among extracellular matrix components is the seed coat epidermal cells of Arabidopsis thaliana. These cells synthesize relatively simple, easily accessible, pectin-rich mucilage that is not essential for plant viability. In this study, we expressed enzymes predicted to modify polysaccharide components of mucilage in the apoplast of seed coat epidermal cells and explored their impacts on mucilage. The seed coat epidermal-specific promoter TESTA ABUNDANT2 (TBA2) was used to drive expression of these enzymes to avoid adverse effects in other parts of the plant. Mature transgenic seeds expressing Rhamnogalacturonate lyase A (RglA) or Rhamnogalacturonate lyase B (RglB) that degrade the pectin rhamnogalacturonan-I (RG-I), a major component of mucilage, had greatly reduced mucilage capsules surrounding the seeds and concomitant decreases in the monosaccharides that comprise the RG-I backbone. Degradation of the minor mucilage component homogalacturonan (HG) using the HG-degrading enzymes Pectin lyase A (PLA) or ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2) resulted in developing seed coat epidermal cells with disrupted cell-cell adhesion and signs of early cell death. These results demonstrate the feasibility of manipulating the seed coat epidermal cell extracellular matrix using a targeted genetic engineering approach.

Characterization of SHATTERPROOF Homoeologs and CRISPR-Cas9-Mediated Genome Editing Enhances Pod-Shattering Resistance in Brassica napus L.

Brassica napus is the most important oil crop plant for edible oil and renewable energy source worldwide. Yield loss caused by pod shattering is a main problem during B. napus harvest. In this study, six BnSHP1 and two BnSHP2 homoeologs were targeted by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) genome editing system and multiple SHP1 and SHP2 mutated lines were identified for evaluating the contribution for pod-shattering resistance. Our data suggest that BnSHP1A09 is probably a promising homoeolog for controlling lignin contents at dehiscence zone. Simultaneous mutation of BnSHP1A09/C04-B/A04 and BnSHP2A05/C04-A exhibited reduced lignified layer and separation layer adjacent to valves and replum. The pod-shattering resistance index (SRI) subsequently increased to 0.31 in five homoeolog mutation lines compared with the wild type (SRI = 0.036), which provide the theoretical basis for breeding of commercial pod-shattering resistance variety.

Pod shattering in grain legumes: emerging genetic and environment-related patterns.

A reduction in pod shattering is one of the main components of grain legume domestication. Despite this, many domesticated legumes suffer serious yield losses due to shattering, particularly under arid conditions. Mutations related to pod shattering modify the twisting force of pod walls or the structural strength of the dehiscence zone in pod sutures. At a molecular level, a growing body of evidence indicates that these changes are controlled by a relatively small number of key genes that have been selected in parallel across grain legume species, supporting partial molecular convergence. Legume homologs of Arabidopsis thaliana silique shattering genes play only minor roles in legume pod shattering. Most domesticated grain legume species contain multiple shattering-resistance genes, with mutants of each gene typically showing only partial shattering resistance. Hence, crosses between varieties with different genes lead to transgressive segregation of shattering alleles, producing plants with either enhanced shattering resistance or atavistic susceptibility to the trait. The frequency of these resistance pod-shattering alleles is often positively correlated with environmental aridity. The continued development of pod-shattering-related functional information will be vital for breeding crops that are suited to the increasingly arid conditions expected in the coming decades.

First approach to pod dehiscence in faba bean: genetic and histological analyses

Pod dehiscence causes important yield losses in cultivated crops and therefore has been a key trait strongly selected against in crop domestication. In spite of the growing knowledge on the genetic basis of dehiscence in different crops, no information is available so far for faba bean. Here we conduct the first comprehensive study for faba bean pod dehiscence by combining, linkage mapping, comparative genomics, QTL analysis and histological examination of mature pods. Mapping of dehiscence-related genes revealed conservation of syntenic blocks among different legumes. Three QTLs were identified in faba bean chromosomes II, IV and VI, although none of them was stable across years. Histological analysis supports the convergent phenotypic evolution previously reported in cereals and related legume species but revealed a more complex pattern in faba bean. Contrary to common bean and soybean, the faba bean dehiscence zone appears to show functional equivalence to that described in crucifers. The lignified wall fiber layer, which is absent in the paucijuga primitive line Vf27, or less lignified and vacuolated in other dehiscent lines, appears to act as the major force triggering pod dehiscence in this species. While our findings, provide new insight into the mechanisms underlying faba bean dehiscence, full understanding of the molecular bases will require further studies combining precise phenotyping with genomic analysis.

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1) releases latent defense signals in stems with reduced lignin content

There is considerable interest in engineering plant cell wall components, particularly lignin, to improve forage quality and biomass properties for processing to fuels and bioproducts. However, modifying lignin content and/or composition in transgenic plants through down-regulation of lignin biosynthetic enzymes can induce expression of defense response genes in the absence of biotic or abiotic stress. Arabidopsis thaliana lines with altered lignin through down-regulation of hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) or loss of function of cinnamoyl CoA reductase 1 (CCR1) express a suite of pathogenesis-related (PR) protein genes. The plants also exhibit extensive cell wall remodeling associated with induction of multiple cell wall-degrading enzymes, a process which renders the corresponding biomass a substrate for growth of the cellulolytic thermophile Caldicellulosiruptor bescii lacking a functional pectinase gene cluster. The cell wall remodeling also results in the release of size- and charge-heterogeneous pectic oligosaccharide elicitors of PR gene expression. Genetic analysis shows that both in planta PR gene expression and release of elicitors are the result of ectopic expression in xylem of the gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1), which is normally expressed during anther and silique dehiscence. These data highlight the importance of pectin in cell wall integrity and the value of lignin modification as a tool to interrogate the informational content of plant cell walls.

CRISPR/Cas9-Mediated Multiplex Genome Editing of JAGGED Gene in Brassica napus L.

Pod shattering resistance is an essential component to achieving a high yield, which is a substantial objective in polyploid rapeseed cultivation. Previous studies have suggested that the Arabidopsis JAGGED (JAG) gene is a key factor implicated in the regulatory web of dehiscence fruit. However, its role in controlling pod shattering resistance in oilseed rape is still unknown. In this study, multiplex genome editing was carried out by the CRISPR/Cas9 system on five homoeologs (BnJAG.A02, BnJAG.C02, BnJAG.C06, BnJAG.A07, and BnJAG.A08) of the JAG gene. Knockout mutagenesis of all homoeologs drastically affected the development of the lateral organs in organizing pod shape and size. The cylindrical body of the pod comprised a number of undifferentiated cells like a callus, without distinctive valves, replum, septum, and valve margins. Pseudoseeds were produced, which were divided into two halves with an incomplete layer of cells (probably septum) that separated the undifferentiated cells. These mutants were not capable of generating any productive seeds for further generations. However, one mutant line was identified in which only a BnJAG.A08-NUB-Like paralog of the JAG gene was mutated. Knockout mutagenesis in BnJAG.A08-NUB gene caused significant changes in the pod dehiscence zone. The replum region of the mutant was increased to a great extent, resulting in enlarged cell size, bumpy fruit, and reduced length compared with the wild type. A higher replum–valve joint area may have increased the resistance to pod shattering by ~2-fold in JAG mutants compared with wild type. Our results offer a basis for understanding variations in Brassica napus fruit by mutating JAG genes and providing a way forward for other Brassicaceae species.

A plant biostimulant from the seaweed Ascophyllum nodosum (Sealicit) reduces podshatter and yield loss in oilseed rape through modulation of IND expression

The yield of podded crops such as oilseed rape (OSR) is limited by evolutionary adaptations of the plants for more efficient and successful seed dispersal for survival. These plants have evolved dehiscent dry fruits that shatter along a specifically developed junction at carpel margins. A number of strategies such as pod sealants, GMOs and hybrids have been developed to mitigate the impact of pod shatter on crop yield with limited success. Plant biostimulants have been shown to influence plant development. A challenge in plant biostimulant research is elucidating the mechanisms of action. Here we have focused on understanding the effect of an Ascophyllum nodosum based biostimulant (Sealicit) on fruit development and seed dispersal trait in Arabidopsis and OSR at genetic and physiological level. The results indicate that Sealicit is affecting the expression of the major regulator of pod shattering, INDEHISCENT, as well as disrupting the auxin minimum. Both factors influence the formation of the dehiscence zone and consequently reduce pod shattering. Unravelling the mode of action of this unique biostimulant provides data to support its effectiveness in reducing pod shatter and highlights its potential for growers to increase seed yield in a number of OSR varieties.

Greater Anatomical Differences of Pod Ventral Suture in Shatter‐Susceptible and Shatter‐Resistant Soybean Cultivars

ABSTRACTFruit dehiscence or seed dispersion is an essential process in the proliferation of wild plants, but it is one of the major factors leading to significant yield losses in domesticated soybean. The ventral suture is critical to pod dehiscence in soybean. To better understand the anatomy of the ventral suture in the soybean pod, we compared the dehiscence zone (DZ) and vascular bundle anatomical structure of the ventral suture using three shatter‐susceptible soybean [Glycine max (L.) Merr.] cultivars and three shatter‐resistant soybean cultivars, which had been evaluated from 140 cultivars over two growing seasons. We found that shatter‐susceptible cultivars, in the cross‐sections of ventral sutures, had a larger vascular bundle area and bundle cap area. The cells of bundle caps in shatter‐susceptible cultivars were even in shape and arranged more closely with less interstitial substance. There was a significantly shorter and straight route from the top of fiber cap cells to the connecting point of the two valves of the pod ventral suture in shatter‐susceptible cultivars than in shatter‐resistant soybean cultivars. Our findings suggest that the route from the top of fiber cap cells to the connecting point of the two valves, the vascular bundle area, the bundle cap area, and the cell arrangement in the bundle cap might be a partial mechanism involved in pod dehiscence of soybean.

Omphalophloios wagneri sp. nov., a new sub-arborescent lycopsid from the middle Moscovian (Middle Pennsylvansian) of the Illinois Basin, USA

Described is a new species of sub-arborescent lycopsid, Omphalophloios wagneri sp. nov. from roof shale of the middle Moscovian (Bolsovian/Atokan) (Middle Pennsylvanian) Lower Block Coal in the Illinois Basin, Indiana, USA. Available material represents mostly fragments of 16 to >60 mm wide fertile axes that bear densely spaced sporangia, which are flattened due to compaction into disc-like shape, 2.5–3.1 mm in diameter. The outer sporangial cell wall layer displays a gradual transition from isometric cells in the central parts of adaxial and abaxial sporangial valves, to a narrow belt of elongated cells in the equatorial area, interpreted as a dehiscence zone. Micro- and megasporangia alternate in irregular patches, a single sporangium being attached adaxially to an extended sporophyll base. Cyperites-like sporophyll distal laminae are ~25 mm long at fertile shoot apices but become progressively longer (>110 mm) down the shoots. Based on size distribution of stems, fertile shoots are estimated to bifurcate at least five times, which suggests that O. wagneri formed a relatively dense crown. The entire habit of the plant is difficult to assess from existing material; however, the tree was probably small and interpreted as colonizing low lying to slightly raised peat swamps. Omphalophloios wagneri differs from all other adpression-based species of the genus in having the smallest sporangia and several orders of fertile-axis bifurcations. In situ megaspores of the Zonalesporites brassertii type also are different from all other Omphalophloios megaspores.

MicroRNA166 Monitors SPOROCYTELESS/NOZZLE for Building of the Anther Internal Boundary.

The internal boundary between inner and outer microsporangia within anthers is essential for male fertility of vascular plants. Dehiscence zones embedded in the boundary release pollen for fertilization. However, the molecular mechanism underlying boundary formation in anthers remains poorly understood. Here, we report that microRNA166 (miR166) and its target PHABULOSA (PHB) regulate SPOROCYTELESS/NOZZLE (SPL/NZZ), which controls microsporogenesis. In developing anthers of Arabidopsis (Arabidopsis thaliana), the expression domains of miR165/6 and SPL/NZZ are overlapped and rearranged synchronously. Dominant mutation of PHB suppresses SPL/NZZ expression on the adaxial sides of stamens, resulting in a thickened boundary, whereas activation of MIR166g up-regulates SPL/NZZ expression, leading to ectopic microsporogenesis in the boundary. PHB limits the expression domains of SPL/NZZ to facilitate construction of the boundary, while miR166 preserves the expression domains of SPL/NZZ by inhibiting PHB to allow the inner microsporangia to take shape. Subsequently, PHB activates the key stem cell maintainer WUSCHEL in anthers to restrict the stomium cells to the boundary so that dehiscence zones develop and release pollen properly. These findings link adaxial/abaxial polarity to microsporogenesis in building of the internal boundary of anthers and thus advance the concepts underlying the establishment of the internal structure of male organs.