Molecular genetic studies suggest that FLORICAULA (FLO)/LEAFY (LFY) orthologs function to control compound leaf development in some legume species. However, loss-of-function mutations in the FLO/LFY orthologs result in reduction of leaf complexity to different degrees in Pisum sativum and Lotus japonicus. To further understand the role of FLO/LFY orthologs in compound leaf development in legumes, we studied compound leaf developmental processes and characterized a leaf development mutant, single leaflet1 (sgl1), from the model legume Medicago truncatula. The sgl1 mutants exhibited strong defects in compound leaf development; all adult leaves in sgl1 mutants are simple due to failure in initiating lateral leaflet primordia. In addition, the sgl1 mutants are also defective in floral development, producing inflorescence-like structures. Molecular cloning of SGL1 revealed that it encodes the M. truncatula FLO/LFY ortholog. When properly expressed, LFY rescued both floral and compound leaf defects of sgl1 mutants, indicating that LFY can functionally substitute SGL1 in compound leaf and floral organ development in M. truncatula. We show that SGL1 and LFY differed in their promoter activities. Although the SGL1 genomic sequence completely rescued floral defects of lfy mutants, it failed to alter the simple leaf structure of the Arabidopsis thaliana plants. Collectively, our data strongly suggest that initiation of lateral leaflet primordia required for compound leaf development involves regulatory processes mediated by the SGL1 function in M. truncatula.

Plant organs, such as seeds, are primary sources of food for both humans and animals. Seed size is one of the major agronomic traits that have been selected in crop plants during their domestication. Legume seeds are a major source of dietary proteins and oils. Here, we report a conserved role for the BIG SEEDS1 (BS1) gene in the control of seed size and weight in the model legume Medicago truncatula and the grain legume soybean (Glycine max). BS1 encodes a plant-specific transcription regulator and plays a key role in the control of the size of plant organs, including seeds, seed pods, and leaves, through a regulatory module that targets primary cell proliferation. Importantly, down-regulation of BS1 orthologs in soybean by an artificial microRNA significantly increased soybean seed size, weight, and amino acid content. Our results provide a strategy for the increase in yield and seed quality in legumes.plant organ size | seed size | forage quality | Medicago | soybean A s a key crop worldwide, soybean not only provides up to 69% of proteins and 30% of oils to the human diet (1) but also requires a low input of fertilizers due to its symbiotic nitrogen fixation ability (2), making it a highly valuable crop to secure global food supplies while contributing to sustainable agriculture. It is clear that the rapid increase of world population requires significant increases in crop production (3).Seed size is a major agronomic trait that has been selected in crop plants during their domestication (4-6). Due to whole genome duplication events that occurred ∼59 and ∼14 Mya (million years ago) (2), soybean has a complex genome structure. Thus, the isolation of key genes or quantitative trait loci (QTL) that control seed size and weight in soybean using conventional approaches can be a challenge and has not been reported to date. To overcome this challenge, we took a molecular genetics approach, using the legume plant Medicago truncatula as a genetic model. We identified BIG SEEDS1 (BS1) as a key gene that controls size of lateral organs, including seeds, seed pods, and leaves, in M. truncatula. Based on these results, we further identified two BS1 orthologs in soybean (Glycine max). Our objectives were to understand the role and regulatory mechanism of the BS1 gene in lateral organ size control in legume plants. Down-regulation of soybean BS1 genes using an artificial microRNA resulted in increased size of seeds, seed pods, and leaves, thus revealing a key and conserved role of BS1 in the control of organ size in legumes. Results and DiscussionIsolation and Characterization of M. truncatula big seeds1 Mutants.In a screen of the fast neutron bombardment (FNB)-induced deletion mutant collection of M. truncatula [cultivar (cv.) Jemalong A17] (7), a unique mutant with large seeds was isolated and named M. truncatula big seeds1-1 (mtbs1-1) (Fig. 1A). The mtbs1-1 mutant also exhibited larger seed pods and leaves than WT plants, suggesting a key role of BS1 in determining lateral organ size (SI Appendix, Figs. S1 and S2). Time...

Plant leaves are diverse in their morphology, reflecting to a large degree the plant diversity in the natural environment. How different leaf morphology is determined is not yet understood. The leguminous plant Medicago truncatula exhibits dissected leaves with three leaflets at the tip. We show that development of the trifoliate leaves is determined by the Cys(2)His(2) zinc finger transcription factor PALM1. Loss-of-function mutants of PALM1 develop dissected leaves with five leaflets clustered at the tip. We demonstrate that PALM1 binds a specific promoter sequence and down-regulates the expression of the M. truncatula LEAFY/ UNIFOLIATA orthologue SINGLE LEAFLET1 (SGL1), encoding an indeterminacy factor necessary for leaflet initiation. Our data indicate that SGL1 is required for leaflet proliferation in the palm1 mutant. Interestingly, ectopic expression of PALM1 effectively suppresses the lobed leaf phenotype from overexpression of a class 1 KNOTTED1-like homeobox protein in Arabidopsis plants. Taken together, our results show that PALM1 acts as a determinacy factor, regulates the spatial-temporal expression of SGL1 during leaf morphogenesis and together with the LEAFY/UNIFOLIATA orthologue plays an important role in orchestrating the compound leaf morphology in M. truncatula.compound leaf development | zinc finger transcription factor PALM1 | LFY/UNI/SGL1 | KNOXI | morphogenesis P lant leaves are lateral organs initiated as a peg-like structure from the flank of the shoot apical meristem (SAM), a pluripotent structure that is capable of self-renewal. They can be simple, consisting of a single flattened blade subtended by a petiole, or compound (or dissected), consisting of multiple blade units known as leaflets. The class 1 Knotted1-like homeobox proteins (KNOXIs) are required to maintain indeterminacy of the SAM (1). During early development, KNOXI genes are downregulated at the incipient leaf primordium at the periphery of the SAM (2). This down-regulation marks the site of primordia initiation and is permanent in developing primordia that lead to simple leaves. However, the KNOXI genes are transiently reactivated in leaf primordia in most eudicot species that have compound leaves, indicating a requirement for a transient phase of indeterminacy in the initiation of leaflet primordia at leaf margins during compound leaf development (3). However, the transient indeterminacy is not sufficient for compound leaf development, as it can also lead to simple leaves as a result of secondary morphogenesis in some plants (3). In some leguminous plants (Fabaceae) that belong to the inverted repeat lacking clade (IRLC), including garden pea (Pisum sativum) and alfalfa (Medicago sativa), the role of KNOXIs in maintaining indeterminacy is replaced by the FLORICAULA (FLO)/LEAFY(LFY) transcription factor UNIFOLIATA (UNI)/ SINGLE LEAFLET1 (SGL1) (4-6), because KNOXI proteins are not detected in leaves in the IRLC legumes (6). However, conflicting evidence exists to support the expression of KNOXI transcripts in leaves of IR...

Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803 adapts to a wide range of adverse environments including high salinity (Joset et al., 1996). These characteristics make it a useful model for understanding stress tolerance in relation to oxygenic photosynthesis. Glucosylglycerol has been identified in Synechocystis sp. PCC 6803 as an osmoprotectant that contributes to its salt tolerance (Mikkat et al., 1996). On the other hand, the mechanisms acting against the ion toxicity of salt stress in the cyanobacterium remain to be elucidated because, not only osmotic pressure, but also ion effects account for its decrease in photosynthetic activity (Allakhverdiev et al., 1999). Na + /H+ antiporter activity appears to be involved in the intracellular Na + homeostasis of Synechococcus sp. PCC 6311 (Blumwald et al., 1984). However, salt stress depresses the activity and synthesis of Na + /H + antiporters in Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 (Allakhverdiev et al., 1999;. There is a dramatic loss of Na+ antiporter activity in Synechocystis sp. PCC 6803 in the presence of 1.0 M NaCl, although it can tolerate up to 1.2 M NaCl (Reed and Stewart, 1985;Allakhverdiev et al., 1999). Apart from Na + /H + antiporters, other membrane transporters may be involved in Na + homeostasis in this cyanobacterium. Both prokaryotic and eukaryotic cells possess primary Na + -ATPases, which extrude Na + directly (Gimmler, 2000). There are several potential Na + -ATPases in the Synechocystis sp. PCC 6803 genome, but no Na + -ATPase in any cyanobacterium has been definitively identified thus far (Ritchie, 1998). Bioenergetic analysis reveals that, under physiological ranges of pH, Na + -coupled secondary ion transport across membranes occurs in Synechococcus sp. PCC 7942 (Ritchie, 1992(Ritchie, , 1998, implying that there is an energyconsuming Na + efflux mechanism in cyanobacteria. Additionally, it has been well established that counteraction of Na + and other metal ions such as K + and Ca 2+ is also a determinant factor in Na + homeostasis. Therefore, it remains to be established whether or not the predicted energy-dependent Na + efflux protein(s) actually exist and the extent to which other cation ATPases may also affect Na + sensitivity of cyanobacteria. In addition to Na + homeostasis, Na + /H + antiporters are also involved in internal pH regulation . In cyanobacteria, light induces the transient acidification of culture medium in a process that /H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728-29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock-outs were evaluated in terms of N...

Medicago truncatula is a legume species belonging to the inverted repeat lacking clade (IRLC) with trifoliolate compound leaves. However, the regulatory mechanisms underlying development of trifoliolate leaves in legumes remain largely unknown. Here, we report isolation and characterization of fused compound leaf1 (fcl1) mutants of M. truncatula. Phenotypic analysis suggests that FCL1 plays a positive role in boundary separation and proximal-distal axis development of compound leaves. Map-based cloning indicates that FCL1 encodes a class M KNOX protein that harbors the MEINOX domain but lacks the homeodomain. Yeast two-hybrid assays show that FCL1 interacts with a subset of Arabidopsis thaliana BEL1-like proteins with slightly different substrate specificities from the Arabidopsis homolog KNATM-B. Double mutant analyses with M. truncatula single leaflet1 (sgl1) and palmate-like pentafoliata1 (palm1) leaf mutants show that fcl1 is epistatic to palm1 and sgl1 is epistatic to fcl1 in terms of leaf complexity and that SGL1 and FCL1 act additively and are required for petiole development. Previous studies have shown that the canonical KNOX proteins are not involved in compound leaf development in IRLC legumes. The identification of FCL1 supports the role of a truncated KNOX protein in compound leaf development in M. truncatula.

An oligohalobic peritrichous ciliate, Epistylis chlorelligerum Shen, 1980, was collected from a ditch in Hangzhou, China. The morphology, oral infraciliature, and morphogenesis of the species were studied using living and protargol-impregnated specimens. Zooids of E. chlorelligerum are 160-230 × 50-60 μm in vivo, and characterized by green-colored endoplasm containing symbiotic algae. The oral infraciliature presents a well-developed filamentous reticulum linked to the circular fiber of the cytostome; the outer two rows of P3 extend adstomally over P1 and usually enfold it. During binary fission, one daughter cell inherits most part of the old buccal apparatus and the reorganized haplokinety and germinal kinety (Hk' and G'), and new buccal apparatus of the other daughter cell is mostly developed from the original germinal kinety (G) and haplokinety (Hk): new peniculi 2, 3 (2P2, 2P3), new haplokinety (2Hk), and new germinal kinety (2G) are formed from G, while the new peniculus 1 (2P1) and its peristomial extention (2Pk) originate from Hk. The epistomial membrane can be observed until the two sets of buccal apparatus begin to separate from each other.

The sugarcane aphid (Melanaphis sacchari) has become a serious pest causing severe economic losses to sorghum [Sorghum bicolor (L.) Moench] grown in the southern United States. Since its original detection in four states in 2013, M. sacchari on sorghum has now, in 2016, spread to 19 states. The presence of one or multiple genotypes on sorghum in the United States has not yet been established. In this study, genome sequencing of M. sacchari was used to develop microsatellite markers. A total of 8,665,267 reads and 1.44 Gb of nucleotide sequences were generated, and 79.6% of the reads were from M. sacchari. Melanaphis sacchari DNA from 46 samples from 17 locations across seven states and one US territory was polymerase chain reaction (PCR) amplified using 38 newly created microsatellite markers, as well as 14 published microsatellite markers. Genotyping with the 52 microsatellite markers indicated that the samples of M. sacchari on sorghum were all one genotype, with the exception of a single sample collected from Sinton, TX, which had the predominant genotype as well as another genotype. Genotyping of the aphid samples with 12 microsatellite markers for Buchnera aphidicola, the obligate aphid symbiont, had nearly identical results. The invasive M. sacchari on sorghum appears to be spreading in the United States on sorghum as primarily one asexual clone.

The process of stomatogenesis in peritrich ciliates is still incompletely understood. Previous studies on the stomatogenesis of four species of peritrichs, Telotrochidium sp., Carchesium polypinum, Opercularia coarctata, and Astylozoon pyriforme conflict with one another in some cases and omit details of events in others. We described the entire process of stomatogenesis in the peritrich ciliate Campanella umbellaria (C. umbellaria) using an improved method of staining with protargol. Our results disagree with some previous studies with regard to the formation of some rudimentary structures, reorganization of the parental haplokinety, formation of new germinal rows, and separation of daughter oral complexes. The pattern of stomatogenesis characteristic of peritrichs is compared to the stomatogenetic patterns of three other oligohymenophorean subclasses and a hypothesis about the evolution of stomatogenesis in the class Oligohymenophorea is offered. Details of stomatogenesis need to be described and verified in a greater variety of peritrichs to clarify possible differences between taxa and make it possible to relate stomatogenesis to evolution within the subclass Peritrichia. Ultrastructural studies are the next step in description of morphogenetic processes in peritrichs, and characteristics of C. umbellaria make it a useful model for this work.