Assessing long-term impacts of cover crops on soil organic carbon in the central US Midwestern agroecosystems.

Cover crops have been reported as one of the most effective practices to increase soil organic carbon (SOC) for agroecosystems. Impacts of cover crops on SOC change vary depending on soil properties, climate, and management practices, but it remains unclear how these control factors affect SOC benefits from cover crops, as well as which management practices can maximize SOC benefits. To address these questions, we used an advanced process-based agroecosystem model, ecosys, to assess the impacts of winter cover cropping on SOC accumulation under different environmental and management conditions. We aimed to answer the following questions: (1) To what extent do cover crops benefit SOC accumulation, and how do SOC benefits from cover crops vary with different factors (i.e., initial soil properties, cover crop types, climate during the cover crop growth period, and cover crop planting and terminating time)? (2) How can we enhance SOC benefits from cover crops under different cover crop management options? Specifically, we first calibrated and validated the ecosys model at two long-term field experiment sites with SOC measurements in Illinois. We then applied the ecosys model to six cover crop field experiment sites spanning across Illinois to assess the impacts of different factors on SOC accumulation. Our modeling results revealed the following findings: (1) Growing cover crops can bring SOC benefits by 0.33 ± 0.06 MgC ha-1  year-1 in six cover crop field experiment sites across Illinois, and the SOC benefits are species specific to legume and non-legume cover crops. (2) Initial SOC stocks and clay contents had overall small influences on SOC benefits from cover crops. During the cover crop growth period (i.e., winter and spring in the US Midwest), high temperature increased SOC benefits from cover crops, while the impacts from larger precipitation on SOC benefits varied field by field. (3) The SOC benefits from cover crops can be maximized by optimizing cover crop management practices (e.g., selecting cover crop types and controlling cover crop growth period) for the US Midwestern maize-soybean rotation system. Finally, we discussed the economic and policy implications of adopting cover crops in the US Midwest, including that current economic incentives to grow cover crops may not be sufficient to cover costs. This study systematically assessed cover crop impacts for SOC change in the US Midwest context, while also demonstrating that the ecosys model, with rigorous validation using field experiment data, can be an effective tool to guide the adaptive management of cover crops and quantify SOC benefits from cover crops. The study thus provides practical tools and insights for practitioners and policy-makers to design cover crop related government agricultural policies and incentive programs for farmers and agri-food related industries.

The Arabidopsis APC4 subunit of the anaphase-promoting complex/cyclosome (APC/C) is critical for both female gametogenesis and embryogenesis.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that is involved in regulating cell-cycle progression. It has been widely studied in yeast and animal cells, but the function and regulation of the APC/C in plant cells are largely unknown. The Arabidopsis APC/C comprises at least 11 subunits, only a few of which have been studied in detail. APC4 is proposed to be a connector in the APC/C in yeast and animals. Here, we report the functional characterization of the Arabidopsis APC4 protein. We examined three heterozygous plant lines carrying apc4 alleles. These plants showed pleiotropic developmental defects in reproductive processes, including abnormal nuclear behavior in the developing embryo sac and aberrant cell division in embryos; these phenotypes differ from those reported for mutants of other subunits. Some ovules and embryos of apc4/+ plants also accumulated cyclin B protein, a known substrate of APC/C, suggesting a compromised function of APC/C. Arabidopsis APC4 was expressed in meristematic cells of seedlings, ovules in pistils and embryos in siliques, and was mainly localized in the nucleus. Additionally, the distribution of auxin was distorted in some embryos of apc4/+ plants. Our results indicate that Arabidopsis APC4 plays critical roles in female gametogenesis and embryogenesis, possibly as a connector in APC/C, and that regulation of auxin distribution may be involved in these processes.

AtMYB14 Regulates Cold Tolerance in Arabidopsis.

Low temperature affects plant growth and crop productivity. The CBF genes are a class of transcription factors that play important roles in cold response. Here we report that AtMYB14 participates in freezing tolerance in Arabidopsis by affecting expression of CBF genes. The AtMYB14 gene was down-regulated by cold treatment. AtMYB14 encodes a nuclear protein that functions as an R2R3-MYB transcription activator. Knock-down of AtMYB14 by artificial microRNA increased the tolerance to freezing stress. Both the CBF genes and the downstream genes were induced to a much higher level in AtMYB14 knock-down plants than in wild type under cold treatment. Our results suggest that AtMYB14 plays an important role in the plant response to cold stress.

The Arabidopsis anaphase-promoting complex/cyclosome subunit 1 is critical for both female gametogenesis and embryogenesis(F).

Anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ligase, plays a critical role in cell cycle control, but the functional characterization of each subunit has not yet been completed. To investigate the function of APC1 in Arabidopsis, we analyzed four mutant alleles of APC1, and found that mutation in APC1 resulted in significantly reduced plant fertility, accumulation of cyclin B, and disrupted auxin distribution in embryos. The three mutant alleles apc1-1, apc1-2 and apc1-3 shared variable defects in female gametogenesis including degradation, abnormal nuclear number, and disrupted polarity of nuclei in the embryo sac as well as in embryogenesis, in which embryos were arrested at multiple stages. All of these defects are similar to those previously identified in apc4. The mutant apc1-4, in which the T-DNA was inserted after the transmembrane domain at the C-terminus, showed much more severe phenotypes; that is, most of the ovules were arrested at the one-nucleate female gametophyte stage (stage FG1). In the apc1 apc4 double mutants, the fertility was further reduced by one-third in apc1-1/+ apc4-1/+, and in some cases no ovules even survived in siliques of apc1-4/+ apc4-1/+. Our data thus suggest that APC1, an essential component of APC/C, plays a synergistic role with APC4 both in female gametogenesis and in embryogenesis.

Arabidopsis RAP2.2 plays an important role in plant resistance to Botrytis cinerea and ethylene responses.

• Ethylene plays a crucial role in plant resistance to necrotrophic pathogens, in which ETHYLENE RESPONSE FACTORs (ERFs) are often involved. • Here, we evaluated the role of an ERF transcription factor, RELATED TO AP2 2 (RAP2.2), in Botrytis resistance and ethylene responses in Arabidopsis. We analyzed the resistance of transgenic plants overexpressing RAP2.2 and the T-DNA insertion mutant to Botrytis cinerea. We assessed its role in the ethylene signaling pathway by molecular and genetic approaches. • RAP2.2-overexpressing transgenic plants showed increased resistance to B. cinerea, whereas its T-DNA insertion mutant rap2.2-3 showed decreased resistance. Overexpression of RAP2.2 in ethylene insensitive 2 (ein2) and ein3 ein3-like 1 (eil1) mutants restored their resistance to B. cinerea. Both ethylene and Botrytis infection induced the expression of RAP2.2 and the induction was disrupted in ein2 and ein3 eil1 mutants. We identified rap2.12-1 as a T-DNA insertion mutant of RAP2.12, the closest homolog of RAP2.2. The hypocotyls of rap2.2-3 rap2.12-1 double mutants showed ethylene insensitivity. The constitutive triple response in constitutive triple response1 (ctr1) was partially released in the rap2.2-3 rap2.12-1 ctr1 triple mutants. • Our findings demonstrate that RAP2.2 functions as an important regulator in Botrytis resistance and ethylene responses.

Solution structure of LCI, a novel antimicrobial peptide from Bacillus subtilis.

LCI, a 47-residue cationic antimicrobial peptide (AMP) found in Bacillus subtilis, is one of the main effective components that have strong antimicrobial activity against Xanthomonas campestris pv Oryzea and Pseudomonas solanacearum PE1, etc. To provide insight into the activity of the peptide, we used nuclear magnetic resonance spectroscopy to determine the structure of recombinant LCI. The solution structure of LCI has a novel topology, containing a four-strand antiparallel ß-sheet as the dominant secondary structure. It is the first structure of the LCI protein family. Different from any known ß-structure AMPs, LCI contains no disulfide bridge or circular structure, suggesting that LCI is also a novel ß-structure AMP.

Over-expression of WOX1 leads to defects in meristem development and polyamine homeostasis in Arabidopsis.

In plants, the meristem has to maintain a separate population of pluripotent cells that serve two main tasks, i.e., self-maintenance and organ initiation, which are separated spatially in meristem. Prior to our study, WUS and WUS-like WOX genes had been reported as essential for the development of the SAM. In this study, the consequences of gain of WOX1 function are described. Here we report the identification of an Arabidopsis gain-of-function mutant wox1-D, in which the expression level of the WOX1 (WUSCHEL HOMEOBOX 1) was elevated and subtle defects in meristem development were observed. The wox1-D mutant phenotype is dwarfed and slightly bushy, with a smaller shoot apex. The wox1-D mutant also produced small and dark green leaves, and exhibited a failure in anther dehiscence and male sterility. Molecular evidences showed that the transcription of the stem cell marker gene CLV3 was down-regulated in the meristem of wox1-D but accumulated in the other regions, i.e., in the root-hypocotyl junction and at the sites for lateral root initiation. The fact that the organ size and cell size in leaves of wox1-D are smaller than those in wild type suggests that cell expansion is possibly affected in order to have partially retarded the development of lateral organs, possibly through alteration of CLV3 expression pattern in the meristem. An S-adenosylmethionine decarboxylase (SAMDC) protein, SAMDC1, was found able to interact with WOX1 by yeast two-hybrid and pull-down assays in vitro. HPLC analysis revealed a significant reduction of polyamine content in wox1-D. Our results suggest that WOX1 plays an important role in meristem development in Arabidopsis, possibly via regulation of SAMDC activity and polyamine homeostasis, and/or by regulating CLV3 expression.

Overexpression of a new putative membrane protein gene AtMRB1 results in organ size enlargement in Arabidopsis.

Arabidopsis AtMRB1 is predicted to encode a novel protein of 432 amino acid residues in length, with four putative trans-membrane domains. In the present study, characterization of AtMRB1 is conducted. Green fluorescent protein (GFP) fusion protein assay showed that AtMRB1 was located in the plasma membrane. Transgenic lines overexpressing AtMRB1 driven by a CaMV 35S promoter were generated. Statistic analysis showed that, during the seedling stage, the organ sizes of the transgenic lines including hypocotyl length, root length and root weight were significantly larger than those of the wild type plants under both light and dark conditions. In the adult plant stage, the AtMRB1 overexpressor plants were found to have larger organ sizes in terms of leaf length and width, and increased number of cauline leaves and branches when bolting. Further observation indicated that the larger leaf size phenotype was due to a larger number of mesophyll cells, the size of which was not altered. Quantitative real-time polymerase chain reaction analysis showed that the transcription of ANT, ROT3 and GRF5 were upregulated in the AtMRB1-overexpressor plants. These data suggest that AtMRB1 is possibly a positive regulator of organ size development in Arabidopsis, mainly through cell number control.

SPOROCYTELESS modulates YUCCA expression to regulate the development of lateral organs in Arabidopsis.

* Auxin is essential for many aspects of plant growth and development, including the determination of lateral organ shapes. * Here, the characterization of a dominant Arabidopsis thaliana mutant spl-D (SPOROCYTELESS dominant), and the roles of SPL in auxin homeostasis and plant development, are reported. * The spl-D mutant displayed a severe up-curling leaf phenotype caused by increased expression of SPOROCYTELESS/NOZZLE (SPL/NZZ), a putative transcription factor gene that was previously linked to sporocyte formation. The spl-D plants also displayed pleiotropic developmental defects including fewer lateral roots, simpler venation patterns, and reduced shoot apical dominance. The leaf and floral phenotypes of spl-D and SPL over-expression lines were reminiscent of yucca (yuc) triple and quadruple mutants, suggesting that SPL may regulate auxin homeostasis. Consistent with this hypothesis, it was found that over-expression of SPL led to down-regulation of the auxin reporter DR5-GUS, and that many auxin-responsive genes were down-regulated in spl-D leaves. Interestingly, the expression of YUC2 and YUC6, two key genes in auxin biosynthesis, was significantly repressed in spl-D plants. * Taken together with the genetic and phenotypic analysis of spl-D/yuc6-D double mutant, these data suggest that SPL may regulate auxin homeostasis by repressing the transcription of YUC2 and YUC6 and participate in lateral organ morphogenesis.

Virus induced gene silencing of AtCDC5 results in accelerated cell death in Arabidopsis leaves.

CDC5, a Myb-related protein, is reported to be essential for the G(2) phase of cell cycle in yeast and animals, but little is known about its function in plants. In this study, Arabidopsis thaliana CDC5 (AtCDC5) is found to be nuclear localized, and the C-terminus of this protein is of transcriptional activation activity in yeast. By taking advantage of the virus induced gene silencing (VIGS) technique, we analyzed the phenotypes of the plants in which AtCDC5 is specifically silenced. The AtCDC5 VIGS plants died before bolting, in which accelerated cell death was detected. Further analysis showed that the transcripts of AtSPT and SAG13, but not SAG12, accumulated in these AtCDC5 VIGS plants, suggesting that the accelerated cell death is different from that occurred during leaf senescence. Furthermore, silencing of AtCDC5 by VIGS in either wild-type, npr1 or nahG plants all induces cell death, suggesting that SA is not crucial for the AtCDC5-associated cell death.

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking.

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem-loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.

A novel bacterium Saprospira sp. strain PdY3 forms bundles and lyses cyanobacteria.

A helical filamentous cyanobactericidal bacterium was isolated from Dianchi Lake, a eutrophic freshwater lake in Kunming City of the Yunnan Province in China using a special solid medium. This species was designated strain PdY3. This bacterium was identified as a novel Saprospira sp. on the basis of its morphological characteristics and 16S rDNA sequence. Strain PdY3 showed apparent group behavior on the solid medium, forming orderly, bundle-like group structures. These bundles moved as groups. Individuals in a bundle responded to the bundle as a whole. PdY3 also showed group behavior and formed a three-dimensional reticular structure when co-cultured with Anabaena in liquid media. This helical bacterium lysed cyanobacteria through direct contact and its group behavior greatly accelerated the cyanobactericidal process. Our experiments showed that PdY3 caused lysis of 64% of Anabaena cells within 1 day and that its cyanobactericidal range was broad. These results underscore potential application of Saprospira on the control of blooms of cyanobacteria. PdY3 group behavior might allow a more efficient capture of bacterial prey.

Enhanced green fluorescence protein tracks trichosanthin in human choriocarcinoma cells as a feasible and stable reporter.

Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) which can inhibit the growth of human choriocarcinoma (JAR) cells. There are no clear mechanisms to discover the interaction pathway and cytotoxicity of TCS in JAR cells. In this paper, we showed the distribution and transport of endogenously expressed TCS in JAR cells. Enhanced Green Fluorescence Protein (EGFP), fused with TCS, was applied as a reporter to track the behavior of TCS in JAR cells. Firstly, we investigated the expression stability of EGFP and physiological effects on JAR cells. A stable cell line expressing EGFP was created, which could reproduce and express EGFP even if transplanted into nude mice. Based on the proved stability and feasibility of EGFP in cultured cells and in vivo, the fusion gene of EGFP and TCS was constructed and transfected into JAR cells by liposome. The fluorescence microscopy showed that TCS-EGFP fusion gene was expressed in JAR cells in 24 to 48 hours and the fluorescence spread in cytoplasm mainly and in nucleus partially, which could trace the distribution and transport of TCS-EGFP in JAR cells. Most of fluorescent cells died after 48 hours for the cytotoxicity of expressed TCS-EGFP. These results first reported a stable expression and tracing method by EGFP in JAR cells, and provided theoretical basis to apply TCS in cancer therapy.

Safety assessment for genetically modified sweet pepper and tomato.

The coat protein (CP) gene of cucumber mosaic virus (CMV) was cloned from a Chinese CMV isolate, the CaMV promoter and NOS terminator added and the gene construct was transformed into both sweet pepper and tomato plants to confer resistance to CMV. Safety assessments of these genetically modified (GM) plants were conducted. It was found that these two GM products showed no genotoxicity either in vitro or in vivo by the micronucleus test, sperm aberration test and Ames test. Animal feeding studies showed no significant differences in growth, body weight gain, food consumption, hematology, blood biochemical indices, organ weights and histopathology between rats or mice of either sex fed with either GM sweet pepper or tomato diets compared with those with non-GM diets. These results demonstrate that the CMV-resistant sweet pepper and tomato are comparable to the non-GM counterparts in terms of food safety.

Sequence-specific assignments of proton NMR resonance peaks and analysis of secondary structural elements of LC1, a novel antibacterial polypeptide.

LC1 is a type of novel antibacterial polypeptide secreted by a Bacillus subtilis strain. It consists of 47 residues. Using bioengineering, LC1 was expressed in E.coli DH5alpha by using recombinant plasmid PBVAB16. By means of two-dimensional DQF-COSY, TOCSY and NOESY spectroscopies, protons of all 47 residues are identified. The studies show that the secondary structures of LC1 are principally anti-parallel beta sheets and extended conformations. It was speculated that there may be a hydrophobic core around Trp(23) in its three-dimensional structure.

Genome sequencing and characterization analysis of a Beijing isolate of chicken coronavirus infectious bronchitis virus.

Avian infectious bronchitis virus (AIBV) is classified as a member of the genus coronavirus in the family coronaviridae. The enveloped virus has a positive-sense, single-stranded RNA genome of approximately 28 kilo-bases, which has a 5' cap structure and 3' polyadenylation tract. The complete genome sequence of infectious bronchitis virus (IBV), Beijing isolate, was determined by cloning sequencing and primer walking. The whole genome is 27733 nucleotides in length, has ten open reading frames: 5' -orf1a-orf1ab-s-3a-3b-e-m-6a-6b-n-3'. Alignments of the genome sequence of IBV Beijing isolate with those of two AIBV strains and one SARS coronavirus were performed respectively. The genome sequence of IBV Beijing isolate compared with that of the IBV strain LX4 (uncompleted, 19440 bp in size) was 91.2% similarity. However, the full-length genome sequence of IBV Beijing isolate was 85.2% identity to that of IBV Strain Beaudette, and was only 50.8% homology to that of SARS coronavirus. The results showed that the genome of IBV has remarkable variation. And IBV Beijing isolate is not closely related to SARS coronavirus. Phylogenetic analyses based on the whole genome sequence, S protein, M protein and N protein, also showed that AIBV Beijing isolate is lone virus in group III and is distant from SARS coronavirus. In conclusion, this study will contribute to the studies of diagnosis and diseases control on IBV in China.

Transcriptional profiling of rice early response to Magnaporthe oryzae identified OsWRKYs as important regulators in rice blast resistance.

Rice blast disease is a major threat to rice production worldwide, but the mechanisms underlying rice resistance to the causal agent Magnaporthe oryzae remain elusive. Therefore, we carried out a transcriptome study on rice early defense response to M. oryzae. We found that the transcriptional profiles of rice compatible and incompatible interactions with M. oryzae were mostly similar, with genes regulated more prominently in the incompatible interactions. The functional analysis showed that the genes involved in signaling and secondary metabolism were extensively up-regulated. In particular, WRKY transcription factor genes were significantly enriched among the up-regulated genes. Overexpressing one of these WRKY genes, OsWRKY47, in transgenic rice plants conferred enhanced resistance against rice blast fungus. Our results revealed the sophisticated transcriptional reprogramming of signaling and metabolic pathways during rice early response to M. oryzae and demonstrated the critical roles of WRKY transcription factors in rice blast resistance.

A nuclear-encoded mitochondrial gene AtCIB22 is essential for plant development in Arabidopsis.

Complex I (the NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain is a complicated, multi-subunit, membrane-bound assembly and contains more than 40 different proteins in higher plants. In this paper, we characterize the Arabidopsis homologue (designated as AtCIB22) of the B22 subunit of eukaryotic mitochondrial Complex I. AtCIB22 is a single-copy gene and is highly conserved throughout eukaryotes. AtCIB22 protein is located in mitochondria and the AtCIB22 gene is widely expressed in different tissues. Mutant Arabidopsis plants with a disrupted AtCIB22 gene display pleiotropic phenotypes including shorter roots, smaller plants and delayed flowering. Stress analysis indicates that the AtCIB22 mutants' seed germination and early seedling growth are severely inhibited by sucrose deprivation stress but more tolerant to ethanol stress. Molecular analysis reveals that in moderate knockdown AtCIB22 mutants, genes including cell redox proteins and stress related proteins are significantly up-regulated, and that in severe knockdown AtCIB22 mutants, the alternative respiratory pathways including NDA1, NDB2, AOX1a and AtPUMP1 are remarkably elevated. These data demonstrate that AtCIB22 is essential for plant development and mitochondrial electron transport chains in Arabidopsis. Our findings also enhance our understanding about the physiological role of Complex I in plants.

A gain-of-function mutation of transcriptional factor PTL results in curly leaves, dwarfism and male sterility by affecting auxin homeostasis.

GT factors are plant-specific trihelix DNA-binding transcription factors, which are involved in light responses and other developmental processes in plant. We identified a gain-of-function mutant of a GT-2 factor gene, PETAL LOSS (PTL), which displayed pleiotropic phenotypes including dwarfism, curly leaves, retarded growth and male sterility. We found that constitutive and ectopic over-expression of PTL driven by the CaMV 35S promoter could not recapitulate the phenotypes of the 35S enhancer-driven mutant ptl-D, and was lethal in some of the transgenic plants at the cotyledon developmental stage, suggesting that accurate temporal and spatial expression of PTL is essential for its proper functional implementation during plant development. Further analysis showed that ptl-D was defective in auxin action and that the alteration of auxin distribution corresponded to the curly leaf phenotype. The fact that degeneration of septum cells and subsequent breakage along the stomium was not observed in ptl-D anthers suggests that defective anther dehiscence was the cause for male sterility. Identification and characterization of the gain-of-function mutant ptl-D will improve our understanding of the diverse functions of GT factors during plant development.

Identification and characterization of COI1-dependent transcription factor genes involved in JA-mediated response to wounding in Arabidopsis plants.

The phytohormone jasmonic acid (JA) is an important signaling molecular involved in many developmental and physiological processes, especially in the response of plants to wounding. In this study, we adopted a new strategy, taking into consideration the microarray data of the CHX treatment, to identify 15 COI1-dependent JA-inducible transcription factors (JCTFs) that have distinct expression patterns in response to wounding. After the analysis on the JCTFs over-expressor plants, we identified four JCTFs, i.e., WRKY18, At1g74930 and At3g53600 in addition to AtMYC2, as the positive regulators in the JA-mediated signaling pathway in response to Arabidopsis wounding.