Regulation of compound leaf development by PHANTASTICA in Medicago truncatula.

Plant leaves, simple or compound, initiate as peg-like structures from the peripheral zone of the shoot apical meristem, which requires class I KNOTTED-LIKE HOMEOBOXI (KNOXI) transcription factors to maintain its activity. The MYB domain protein encoded by the ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) gene, together with other factors, excludes KNOXI gene expression from incipient leaf primordia to initiate leaves and specify leaf adaxial identity. However, the regulatory relationship between ARP and KNOXI is more complex in compound-leafed species. Here, we investigated the role of ARP and KNOXI genes in compound leaf development in Medicago truncatula. We show that the M. truncatula phantastica mutant exhibited severe compound leaf defects, including curling and deep serration of leaf margins, shortened petioles, increased rachises, petioles acquiring motor organ characteristics, and ectopic development of petiolules. On the other hand, the M. truncatula brevipedicellus mutant did not exhibit visible compound leaf defects. Our analyses show that the altered petiole development requires ectopic expression of ELONGATED PETIOLULE1, which encodes a lateral organ boundary domain protein, and that the distal margin serration requires the auxin efflux protein M. truncatula PIN-FORMED10 in the M. truncatula phantastica mutant.

Strigolactones contribute to shoot elongation and to the formation of leaf margin serrations in Medicago truncatula R108.

Strigolactones were recently identified as a new class of plant hormones involved in the control of shoot branching. The characterization of strigolactone mutants in several species has progressively revealed their contribution to several other aspects of development in roots and shoots. In this article, we characterize strigolactone-deficient and strigolactone-insensitive mutants of the model legume Medicago truncatula for aerial developmental traits. The most striking mutant phenotype observed was compact shoot architecture. In contrast with what was reported in other species, this could not be attributed to enhanced shoot branching, but was instead due to reduced shoot elongation. Another notable feature was the modified leaf shape in strigolactone mutants: serrations at the leaf margin were smaller in the mutants than in wild-type plants. This phenotype could be rescued in a dose-dependent manner by exogenous strigolactone treatments of strigolactone-deficient mutants, but not of strigolactone-insensitive mutants. Treatment with the auxin transport inhibitor N-1-naphthylphtalamic acid resulted in smooth leaf margins, opposite to the effect of strigolactone treatment. The contribution of strigolactones to the formation of leaf serrations in M. truncatula R108 line represents a novel function of these hormones, which has not been revealed by the analysis of strigolactone mutants in other species.

Regulation of compound leaf development in Medicago truncatula by fused compound leaf1, a class M KNOX gene.

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.

Functional remodeling of gut microbiota and liver in laying hens as affected by fasting and refeeding after fasting.

Objective: Animals will experience energy deprivation processes such as moulting, clutching, migration and long-distance transportation under natural survival conditions and in production practices, and the body will trigger a series of adaptive metabolic changes during these processes. Fasting and refeeding after fasting can induce remodeling of nutrients and energy metabolism. This study aims to investigate the mechanisms by which the gut microbiota and liver of poultry respond to energy deprivation under specific conditions. Methods: Ninety 252-day-old laying hens were randomly divided into 3 groups: (1) fed ad libitum (control group); (2) fasted from day 13 to day 17 (fasting group); (3) fasted from day 1 to day 5, then refed on a specific feeding way (refeeding group). After that, the serum, liver, jejunum tissues, and cecum contents were sampled and sent for metabolome, transcriptome, morphology, and 16S rDNA sequencing analyses, respectively. Results: Results showed that food deprivation not only observably decreased the body weight, liver index, and the villus height and villus/crypt ratio of jejunum, but also significantly changed the gut microbiota compositions, serum metabolic profiles, and the hepatic gene expression patterns of laying hens, whereas these changes were effectively reversed by the following refeeding operation. At the same time, metabolome combined transcriptome analysis revealed that both serum differential metabolites and hepatic differential expressed genes (DEGs) were consistently enriched in the lipid and amino metabolism pathways, and strong correlations were synchronously found between the differential metabolites and both of the differential gut microbial genera and DEGs, suggesting the crosstalks among gut, liver and their resulting serum metabolic products. Conclusion: The results suggested that the organism might coordinate to maintain metabolic homeostasis under energy deprivation through a combination of changes in gut microbial composition and hepatic gene expression.

Xanthomonas campestris VemR enhances the transcription of the T3SS key regulator HrpX via physical interaction with HrpG.

VemR is a response regulator of the two-component signalling systems (TCSs). It consists solely of a receiver domain. Previous studies have shown that VemR plays an important role in influencing the production of exopolysaccharides and exoenzymes, cell motility, and virulence of Xanthomonas campestris pv. campestris (Xcc). However, whether VemR is involved in the essential pathogenicity determinant type III secretion system (T3SS) is unclear. In this work, we found by transcriptome analysis that VemR modulates about 10% of Xcc genes, which are involved in various cellular processes including the T3SS. Further experiments revealed that VemR physically interacts with numerous proteins, including the TCS sensor kinases HpaS and RavA, and the TCS response regulator HrpG, which directly activates the transcription of HrpX, a key regulator controlling T3SS expression. It has been demonstrated previously that HpaS composes a TCS with HrpG or VemR to control the expression of T3SS or swimming motility, while RavA and VemR form a TCS to control the expression of flagellar genes. Mutation analysis and in vitro transcription assay revealed that phosphorylation might be essential for the function of VemR and phosphorylated VemR could significantly enhance the activation of hrpX transcription by HrpG. We infer that the binding of VemR to HrpG can modulate the activity of HrpG to the hrpX promoter, thereby enhancing hrpX transcription. Although further studies are required to validate this inference and explore the detailed functional mechanism of VemR, our findings provide some insights into the complex regulatory cascade of the HpaS/RavA-VemR/HrpG-HrpX signal transduction system in the control of T3SS.

NO APICAL MERISTEM (MtNAM) regulates floral organ identity and lateral organ separation in Medicago truncatula.

• The CUP-SHAPED COTYLEDON (CUC)/NO APICAL MERISTEM (NAM) family of genes control boundary formation and lateral organ separation, which is critical for proper leaf and flower patterning. However, most downstream targets of CUC/NAM genes remain unclear. • In a forward screen of the tobacco retrotransposon1 (Tnt1) insertion population in Medicago truncatula, we isolated a weak allele of the no-apical-meristem mutant mtnam-2. Meanwhile, we regenerated a mature plant from the null allele mtnam-1. These materials allowed us to extensively characterize the function of MtNAM and its downstream genes. • MtNAM is highly expressed in vegetative shoot buds and inflorescence apices, specifically at boundaries between the shoot apical meristem and leaf/flower primordia. Mature plants of the regenerated null allele and the weak allele display remarkable floral phenotypes: floral whorls and organ numbers are reduced and the floral organ identity is compromised. Microarray and quantitative RT-PCR analyses revealed that all classes of floral homeotic genes are down-regulated in mtnam mutants. Mutations in MtNAM also lead to fused cotyledons and leaflets of the compound leaf as well as a defective shoot apical meristem. • Our results revealed that MtNAM shares the role of CUC/NAM family genes in lateral organ separation and compound leaf development, and is also required for floral organ identity and development.

AtRabD2b and AtRabD2c have overlapping functions in pollen development and pollen tube growth.

BACKGROUND: Rab GTPases are important regulators of endomembrane trafficking, regulating exocytosis, endocytosis and membrane recycling. Many Rab-like proteins exist in plants, but only a subset have been functionally characterized. RESULTS: Here we report that AtRabD2b and AtRabD2c play important roles in pollen development, germination and tube elongation. AtrabD2b and AtrabD2c single mutants have no obvious morphological changes compared with wild-type plants across a variety of growth conditions. An AtrabD2b/2c double mutant is also indistinguishable from wild-type plants during vegetative growth; however its siliques are shorter than those in wild-type plants. Compared with wild-type plants, AtrabD2b/2c mutants produce deformed pollen with swollen and branched pollen tube tips. The shorter siliques in the AtrabD2b/2c double mutant were found to be primarily due to the pollen defects. AtRabD2b and AtRabD2c have different but overlapping expression patterns, and they are both highly expressed in pollen. Both AtRabD2b and AtRabD2c protein localize to Golgi bodies. CONCLUSIONS: These findings support a partially redundant role for AtRabD2b and AtRabD2c in vesicle trafficking during pollen tube growth that cannot be fulfilled by the remaining AtRabD family members.

High through-put determination of 28 veterinary antibiotic residues in swine wastewater by one-step dispersive solid phase extraction sample cleanup coupled with ultra-performance liquid chromatography-tandem mass spectrometry.

We developed a QuEChERS (quick, easy, cheap, effective, rugged and safe) method for the high through-put determination of 28 common veterinary antibiotics in swine wastewater using one-step dispersive solid-phase extraction (d-SPE) for sample cleanup and ultra-performance liquid chromatography-tandem mass spectrometry for detection. The orthogonal test method was used to systematically investigate the parameters that might influence d-SPE efficiency. The optimal d-SPE procedure utilized 40 mg primary secondary amine sorbent and 3 g L-1 Na2EDTA. The recoveries ranged from 50 to 100% with relative standard deviations <20% for all target analytes except for enrofloxacin and chlortetracycline. The limits of detection and limits of quantification for all the analytes ranged from 0.002 to 0.200 ng mL-1 and 0.005-0.500 ng mL-1, respectively. The developed method was successfully applied to the analysis of 28 antibiotic residues in swine wastewater from 10 pig farms located in central China. Fourteen antibiotics including 4 sulfonamides (sulfadiazine, sulfamerazine, sulfamonomethoxine and trimethoprim), 5 fluoroquinolones (norfloxacin, ciprofloxacin, pefloxacin, enrofloxacin, and ofloxacin), 1 lincosamide (lincomycin) and 4 tetracyclines (doxycycline, tetracycline, oxytetracycline, and chlortetracycline) were detected at levels ranging from 0.0560 to 1793 ng mL-1. Our results demonstrated that the optimized method is a simple but reliable analytical technique for the routine monitoring of veterinary antibiotics in swine wastewater. Swine wastewater samples that we analyzed from 10 pig farms in Jiangxi Province, China were highly contaminated and pose a serious threat to ecosystems and to public health.

Articulation of three core metabolic processes in Arabidopsis: fatty acid biosynthesis, leucine catabolism and starch metabolism.

BACKGROUND: Elucidating metabolic network structures and functions in multicellular organisms is an emerging goal of functional genomics. We describe the co-expression network of three core metabolic processes in the genetic model plant Arabidopsis thaliana: fatty acid biosynthesis, starch metabolism and amino acid (leucine) catabolism. RESULTS: These co-expression networks form modules populated by genes coding for enzymes that represent the reactions generally considered to define each pathway. However, the modules also incorporate a wider set of genes that encode transporters, cofactor biosynthetic enzymes, precursor-producing enzymes, and regulatory molecules. We tested experimentally the hypothesis that one of the genes tightly co-expressed with starch metabolism module, a putative kinase AtPERK10, will have a role in this process. Indeed, knockout lines of AtPERK10 have an altered starch accumulation. In addition, the co-expression data define a novel hierarchical transcript-level structure associated with catabolism, in which genes performing smaller, more specific tasks appear to be recruited into higher-order modules with a broader catabolic function. CONCLUSION: Each of these core metabolic pathways is structured as a module of co-expressed transcripts that co-accumulate over a wide range of environmental and genetic perturbations and developmental stages, and represent an expanded set of macromolecules associated with the common task of supporting the functionality of each metabolic pathway. As experimentally demonstrated, co-expression analysis can provide a rich approach towards understanding gene function.

LeafletAnalyzer, an Automated Software for Quantifying, Comparing and Classifying Blade and Serration Features of Compound Leaves during Development, and among Induced Mutants and Natural Variants in the Legume Medicago truncatula.

Diverse leaf forms ranging from simple to compound leaves are found in plants. It is known that the final leaf size and shape vary greatly in response to developmental and environmental changes. However, changes in leaf size and shape have been quantitatively characterized only in a limited number of species. Here, we report development of LeafletAnalyzer, an automated image analysis and classification software to analyze and classify blade and serration characteristics of trifoliate leaves in Medicago truncatula. The software processes high quality leaf images in an automated or manual fashion to generate size and shape parameters for both blades and serrations. In addition, it generates spectral components for each leaflets using elliptic Fourier transformation. Reconstruction studies show that the spectral components can be reliably used to rebuild the original leaflet images, with low, and middle and high frequency spectral components corresponding to the outline and serration of leaflets, respectively. The software uses artificial neutral network or k-means classification method to classify leaflet groups that are developed either on successive nodes of stems within a genotype or among genotypes such as natural variants and developmental mutants. The automated feature of the software allows analysis of thousands of leaf samples within a short period of time, thus facilitating identification, comparison and classification of leaf groups based on leaflet size, shape and tooth features during leaf development, and among induced mutants and natural variants.

AUXIN RESPONSE FACTOR3 Regulates Compound Leaf Patterning by Directly Repressing PALMATE-LIKE PENTAFOLIATA1 Expression in Medicago truncatula.

Diverse leaf forms can be seen in nature. In Medicago truncatula, PALM1 encoding a Cys(2)His(2) transcription factor is a key regulator of compound leaf patterning. PALM1 negatively regulates expression of SGL1, a key regulator of lateral leaflet initiation. However, how PALM1 itself is regulated is not yet known. To answer this question, we used promoter sequence analysis, yeast one-hybrid tests, quantitative transcription activity assays, ChIP-PCR analysis, and phenotypic analyses of overexpression lines and mutant plants. The results show that M. truncatula AUXIN RESPONSE FACTOR3 (MtARF3) functions as a direct transcriptional repressor of PALM1. MtARF3 physically binds to the PALM1 promoter sequence in yeast cells. MtARF3 selectively interacts with specific auxin response elements (AuxREs) in the PALM1 promoter to repress reporter gene expression in tobacco leaves and binds to specific sequences in the PALM1 promoter in vivo. Upregulation of MtARF3 or removal of both PHANTASTICA (PHAN) and ARGONAUTE7 (AGO7) pathways resulted in compound leaves with five narrow leaflets arranged in a palmate-like configuration. These results support that MtARF3, in addition as an adaxial-abaxial polarity regulator, functions to restrict spatiotemporal expression of PALM1, linking auxin signaling to compound leaf patterning in the legume plant M. truncatula.

Expression of harpin(xoo) in transgenic tobacco induces pathogen defense in the absence of hypersensitive cell death.

ABSTRACT Harpin(Xoo), encoded by the hpaG(Xoo) gene of Xanthomonas oryzae pv. oryzae, is a member of the harpin group of proteins that induce pathogen resistance and hypersensitive cell death (HCD) in plants. We elaborated whether both processes are correlated in hpaG(Xoo)-expressing tobacco (HARTOB) plants, which produced harpin(Xoo) intracellularly. Resistance to fungal, bacterial, and viral pathogens increased in HARTOB, in correlation with the expression of hpaG(Xoo), the gene NPR1 that regulates several resistance pathways, and defense genes GST1, Chia5, PR-1a, and PR-1b that are mediated by different signals. However, reactive oxygen intermediate burst, the expression of HCD marker genes hsr203 and hin1, and cell death did not occur spontaneously in HARTOB, though they did in untransformed and HARTOB plants treated exogenously with harpin(Xoo). Thus, the transgenic expression of harpin(Xoo) confers nonspecific pathogen defense in the absence of HCD.

Auxin efflux transporter MtPIN10 regulates compound leaf and flower development in Medicago truncatula.

Plant diversity in nature is to a large extent reflected by morphological diversity of their leaves. Both simple and dissected (with multiple blades or leaflets) leaves are initiated from shoot apical meristem (SAM) in a highly ordered fashion. Similarly, development of leaflets from leaf marginal meristem (marginal blastozone) is also highly ordered. How morphological diversity of plant leaves is regulated remains an important topic of studies on plant form evolution. Here, we describe isolation and characterization of loss-of-function mutants of auxin efflux transporter MtPIN10 of a legume species, Medicago truncatula. Mtpin10 mutants exhibit defects in diverse developmental processes including leaf and leaflet development. Cross species genetic complementation demonstrates that MtPIN10 and Arabidopsis PIN1 are functional orthologs. Double mutant analyses reveal complex genetic interactions between MtPIN10 and Medicago SINGLE LEAFLET1 (SGL1), and CUP-SHAPED COTYLEDON2 (MtCUC2), three regulatory genes involved in developmental processes including dissected leaf and flower development. 

The E3 ubiquitin ligase SCFTIR1/AFB and membrane sterols play key roles in auxin regulation of endocytosis, recycling, and plasma membrane accumulation of the auxin efflux transporter PIN2 in Arabidopsis thaliana.

The PIN family of auxin efflux transporters exhibit polar plasma membrane (PM) localization and play a key role in auxin gradient-mediated developmental processes. Auxin inhibits PIN2 endocytosis and promotes its PM localization. However, the underlying mechanisms remain elusive. Here, we show that the inhibitory effect of auxin on PIN2 endocytosis was impaired in SCF(TIR1/AFB) auxin signaling mutants. Similarly, reducing membrane sterols impaired auxin inhibition of PIN2 endocytosis. Gas chromatography-mass spectrometry analyses indicate that membrane sterols were significantly reduced in SCF(TIR1/AFB) mutants, supporting a link between membrane sterols and auxin signaling in regulating PIN2 endocytosis. We show that auxin promoted PIN2 recycling from endosomes to the PM and increased PIN2 steady state levels in the PM fraction. Furthermore, we show that the positive effect of auxin on PIN2 levels in the PM was impaired by inhibiting membrane sterols or auxin signaling. Consistent with this, the sterol biosynthetic mutant fk-J79 exhibited pronounced defects in primary root elongation and gravitropic response. Our data collectively indicate that, although there are distinct processes involved in endocytic regulation of specific PM-resident proteins, the SCF(TIR1/AFB)-dependent processes are required for auxin regulation of endocytosis, recycling, and PM accumulation of the auxin efflux transporter PIN2 in Arabidopsis thaliana.

The ABI2-dependent abscisic acid signalling controls HrpN-induced drought tolerance in Arabidopsis.

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.

Downstream divergence of the ethylene signaling pathway for harpin-stimulated Arabidopsis growth and insect defense.

Ethylene (ET) signal transduction may regulate plant growth and defense, depending on which components are recruited into the pathway in response to different stimuli. We report here that the ET pathway controls both insect resistance (IR) and plant growth enhancement (PGE) in Arabidopsis (Arabidopsis thaliana) plants responding to harpin, a protein produced by a plant pathogenic bacterium. PGE may result from spraying plant tops with harpin or by soaking seeds in harpin solution; the latter especially enhances root growth. Plants treated similarly develop resistance to the green peach aphid (Myzus persicae). The salicylic acid pathway, although activated by harpin, does not lead to PGE and IR. By contrast, PGE and IR are induced in both wild-type plants and genotypes that have defects in salicylic acid signaling. In response to harpin, levels of jasmonic acid (JA) decrease, and the COI1 gene, which is indispensable for JA signal transduction, is not expressed in wild-type plants. However, PGE and IR are stimulated in the JA-resistant mutant jar1-1. In the wild type, PGE and IR develop coincidently with increases in ET levels and the expression of several genes essential for ET signaling. The ET receptor gene ETR1 is required because both phenotypes are arrested in the etr1-1 mutant. Consistently, inhibition of ET perception nullifies the induction of both PGE and IR. The signal transducer EIN2 is required for IR, and EIN5 is required for PGE because IR and PGE are impaired correspondingly in the ein2-1 and ein5-1 mutants. Therefore, harpin activates ET signaling while conscribing EIN2 and EIN5 to confer IR and PGE, respectively.