China Agricultural University

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Cropland Acidification in China China is experiencing increasing problems with acid rain, groundwater pollution, and nitrous oxide emissions. Rapid development of industry and transportation has accelerated nitrate (N) emissions to the atmosphere. Consequently, soil degradation, water shortage, and pollution, in addition to atmospheric quality decline are becoming major public concerns across China. Since the 1990s, China has become both the largest consumer of chemical N fertilizers and the highest cereal producer in the world, which has consequences for arable soil acidification. Guo et al. (p. 1008 , published online 11 February) present a meta-analysis of a regional acidification phenomenon in Chinese arable soils that is largely associated with higher N fertilization and higher crop production. Such large-scale soil acidification is likely to threaten the sustainability of agriculture and affect the biogeochemical cycles of nutrients and also toxic elements in soils.

This paper reports the genome sequence of domesticated tomato, a major crop plant, and a draft sequence for its closest wild relative; comparative genomics reveal very little divergence between the two genomes but some important differences with the potato genome, another important food crop in the genus Solanum. Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera1 and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium2, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

The agriGO platform, which has been serving the scientific community for >10 years, specifically focuses on gene ontology (GO) enrichment analyses of plant and agricultural species. We continuously maintain and update the databases and accommodate the various requests of our global users. Here, we present our updated agriGO that has a largely expanded number of supporting species (394) and datatypes (865). In addition, a larger number of species have been classified into groups covering crops, vegetables, fish, birds and insects closely related to the agricultural community. We further improved the computational efficiency, including the batch analysis and P-value distribution (PVD), and the user-friendliness of the web pages. More visualization features were added to the platform, including SEACOMPARE (cross comparison of singular enrichment analysis), direct acyclic graph (DAG) and Scatter Plots, which can be merged by choosing any significant GO term. The updated platform agriGO v2.0 is now publicly accessible at http://systemsbiology.cau.edu.cn/agriGOv2/.

Excessive N fertilization in intensive agricultural areas of China has resulted in serious environmental problems because of atmospheric, soil, and water enrichment with reactive N of agricultural origin. This study examines grain yields and N loss pathways using a synthetic approach in 2 of the most intensive double-cropping systems in China: waterlogged rice/upland wheat in the Taihu region of east China versus irrigated wheat/rainfed maize on the North China Plain. When compared with knowledge-based optimum N fertilization with 30-60% N savings, we found that current agricultural N practices with 550-600 kg of N per hectare fertilizer annually do not significantly increase crop yields but do lead to about 2 times larger N losses to the environment. The higher N loss rates and lower N retention rates indicate little utilization of residual N by the succeeding crop in rice/wheat systems in comparison with wheat/maize systems. Periodic waterlogging of upland systems caused large N losses by denitrification in the Taihu region. Calcareous soils and concentrated summer rainfall resulted in ammonia volatilization (19% for wheat and 24% for maize) and nitrate leaching being the main N loss pathways in wheat/maize systems. More than 2-fold increases in atmospheric deposition and irrigation water N reflect heavy air and water pollution and these have become important N sources to agricultural ecosystems. A better N balance can be achieved without sacrificing crop yields but significantly reducing environmental risk by adopting optimum N fertilization techniques, controlling the primary N loss pathways, and improving the performance of the agricultural Extension Service.

Gene Ontology (GO), the de facto standard in gene functionality description, is used widely in functional annotation and enrichment analysis. Here, we introduce agriGO, an integrated web-based GO analysis toolkit for the agricultural community, using the advantages of our previous GO enrichment tool (EasyGO), to meet analysis demands from new technologies and research objectives. EasyGO is valuable for its proficiency, and has proved useful in uncovering biological knowledge in massive data sets from high-throughput experiments. For agriGO, the system architecture and website interface were redesigned to improve performance and accessibility. The supported organisms and gene identifiers were substantially expanded (including 38 agricultural species composed of 274 data types). The requirement on user input is more flexible, in that user-defined reference and annotation are accepted. Moreover, a new analysis approach using Gene Set Enrichment Analysis strategy and customizable features is provided. Four tools, SEA (Singular enrichment analysis), PAGE (Parametric Analysis of Gene set Enrichment), BLAST4ID (Transfer IDs by BLAST) and SEACOMPARE (Cross comparison of SEA), are integrated as a toolkit to meet different demands. We also provide a cross-comparison service so that different data sets can be compared and explored in a visualized way. Lastly, agriGO functions as a GO data repository with search and download functions; agriGO is publicly accessible at http://bioinfo.cau.edu.cn/agriGO/.

With the goal of providing a comprehensive, high-quality resource for both plant transcription factors (TFs) and their regulatory interactions with target genes, we upgraded plant TF database PlantTFDB to version 4.0 (http://planttfdb.cbi.pku.edu.cn/). In the new version, we identified 320 370 TFs from 165 species, presenting a more comprehensive genomic TF repertoires of green plants. Besides updating the pre-existing abundant functional and evolutionary annotation for identified TFs, we generated three new types of annotation which provide more directly clues to investigate functional mechanisms underlying: (i) a set of high-quality, non-redundant TF binding motifs derived from experiments; (ii) multiple types of regulatory elements identified from high-throughput sequencing data; (iii) regulatory interactions curated from literature and inferred by combining TF binding motifs and regulatory elements. In addition, we upgraded previous TF prediction server, and set up four novel tools for regulation prediction and functional enrichment analyses. Finally, we set up a novel companion portal PlantRegMap (http://plantregmap.cbi.pku.edu.cn) for users to access the regulation resource and analysis tools conveniently.

This study—based on systematic testing of 30 different wheat crop models against field experiments—shows that many wheat models simulate yields well, but with reduced accuracy at higher temperatures. Extrapolation of the model ensemble response indicates that global wheat production will fall by 6% for each 1 °C increase in temperature. Crop models are essential tools for assessing the threat of climate change to local and global food production1. Present models used to predict wheat grain yield are highly uncertain when simulating how crops respond to temperature2. Here we systematically tested 30 different wheat crop models of the Agricultural Model Intercomparison and Improvement Project against field experiments in which growing season mean temperatures ranged from 15 °C to 32 °C, including experiments with artificial heating. Many models simulated yields well, but were less accurate at higher temperatures. The model ensemble median was consistently more accurate in simulating the crop temperature response than any single model, regardless of the input information used. Extrapolating the model ensemble temperature response indicates that warming is already slowing yield gains at a majority of wheat-growing locations. Global wheat production is estimated to fall by 6% for each °C of further temperature increase and become more variable over space and time.

Reactive oxygen species (ROS) are produced by living cells as normal cellular metabolic byproduct. Under excessive stress conditions, cells will produce numerous ROS, and the living organisms eventually evolve series of response mechanisms to adapt to the ROS exposure as well as utilize it as the signaling molecules. ROS molecules would trigger oxidative stress in a feedback mechanism involving many biological processes, such as apoptosis, necrosis and autophagy. Growing evidences have suggested that ROS play a critical role as the signaling molecules throughout the entire cell death pathway. Overwhelming production of ROS can destroy organelles structure and bio-molecules, which lead to inflammatory response that is a known underpinning mechanism for the development of diabetes and cancer. Cytochrome P450 enzymes (CYP) are regarded as the markers of oxidative stress, can transform toxic metabolites into ROS, such as superoxide anion, hydrogen peroxide and hydroxyl radical which might cause injury of cells. Accordingly, cells have evolved a balanced system to neutralize the extra ROS, namely antioxidant systems that consist of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidases (GPxs), thioredoxin (Trx) as well as the non-enzymatic antioxidants which collectively reduce oxidative state. Herein, we review the recent novel findings of cellular processes induced by ROS, and summarize the roles of cellular endogenous antioxidant systems as well as natural anti-oxidative compounds in several human diseases caused by ROS in order to illustrate the vital role of antioxidants in prevention against oxidative stress.

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives.

To better determine the history of modern birds, we performed a genome-scale phylogenetic analysis of 48 species representing all orders of Neoaves using phylogenomic methods created to handle genome-scale data. We recovered a highly resolved tree that confirms previously controversial sister or close relationships. We identified the first divergence in Neoaves, two groups we named Passerea and Columbea, representing independent lineages of diverse and convergently evolved land and water bird species. Among Passerea, we infer the common ancestor of core landbirds to have been an apex predator and confirm independent gains of vocal learning. Among Columbea, we identify pigeons and flamingoes as belonging to sister clades. Even with whole genomes, some of the earliest branches in Neoaves proved challenging to resolve, which was best explained by massive protein-coding sequence convergence and high levels of incomplete lineage sorting that occurred during a rapid radiation after the Cretaceous-Paleogene mass extinction event about 66 million years ago.

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.

With increasing demand of agricultural production and as the peak in global production will occur in the next decades, phosphorus (P) is receiving more attention as a nonrenewable resource ([Cordell et al., 2009][1]; [Gilbert, 2009][2]). One unique characteristic of P is its low availability due to

BACKGROUND: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. RESULTS: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. CONCLUSIONS: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Contents Summary 523 I. Introduction 523 II. Sensing salt stress 524 III. Ion homeostasis regulation 524 IV. Metabolite and cell activity responses to salt stress 527 V. Conclusions and perspectives 532 Acknowledgements 533 References 533 SUMMARY: Excess soluble salts in soil (saline soils) are harmful to most plants. Salt imposes osmotic, ionic, and secondary stresses on plants. Over the past two decades, many determinants of salt tolerance and their regulatory mechanisms have been identified and characterized using molecular genetics and genomics approaches. This review describes recent progress in deciphering the mechanisms controlling ion homeostasis, cell activity responses, and epigenetic regulation in plants under salt stress. Finally, we highlight research areas that require further research to reveal new determinants of salt tolerance in plants.

A deficiency of dietary protein or amino acids has long been known to impair immune function and increase the susceptibility of animals and humans to infectious disease. However, only in the past 15 years have the underlying cellular and molecular mechanisms begun to unfold. Protein malnutrition reduces concentrations of most amino acids in plasma. Findings from recent studies indicate an important role for amino acids in immune responses by regulating: (1) the activation of T lymphocytes, B lymphocytes, natural killer cells and macrophages; (2) cellular redox state, gene expression and lymphocyte proliferation; and (3) the production of antibodies, cytokines and other cytotoxic substances. Increasing evidence shows that dietary supplementation of specific amino acids to animals and humans with malnutrition and infectious disease enhances the immune status, thereby reducing morbidity and mortality. Arginine, glutamine and cysteine precursors are the best prototypes. Because of a negative impact of imbalance and antagonism among amino acids on nutrient intake and utilisation, care should be exercised in developing effective strategies of enteral or parenteral provision for maximum health benefits. Such measures should be based on knowledge about the biochemistry and physiology of amino acids, their roles in immune responses, nutritional and pathological states of individuals and expected treatment outcomes. New knowledge about the metabolism of amino acids in leucocytes is critical for the development of effective means to prevent and treat immunodeficient diseases. These nutrients hold great promise in improving health and preventing infectious diseases in animals and humans.

Nutrient additions to intensive agricultural systems range from inadequate to excessive—and both extremes have substantial human and environmental costs.

Plants transport fixed carbon predominantly as sucrose, which is produced in mesophyll cells and imported into phloem cells for translocation throughout the plant. It is not known how sucrose migrates from sites of synthesis in the mesophyll to the phloem, or which cells mediate efflux into the apoplasm as a prerequisite for phloem loading by the SUT sucrose-H(+) (proton) cotransporters. Using optical sucrose sensors, we identified a subfamily of SWEET sucrose efflux transporters. AtSWEET11 and 12 localize to the plasma membrane of the phloem. Mutant plants carrying insertions in AtSWEET11 and 12 are defective in phloem loading, thus revealing a two-step mechanism of SWEET-mediated export from parenchyma cells feeding H(+)-coupled import into the sieve element-companion cell complex. We discuss how restriction of intercellular transport to the interface of adjacent phloem cells may be an effective mechanism to limit the availability of photosynthetic carbon in the leaf apoplasm in order to prevent pathogen infections.

Intercropping is a farming practice involving two or more crop species, or genotypes, growing together and coexisting for a time. On the fringes of modern intensive agriculture, intercropping is important in many subsistence or low-input/resource-limited agricultural systems. By allowing genuine yield gains without increased inputs, or greater stability of yield with decreased inputs, intercropping could be one route to delivering ‘sustainable intensification’. We discuss how recent knowledge from agronomy, plant physiology and ecology can be combined with the aim of improving intercropping systems. Recent advances in agronomy and plant physiology include better understanding of the mechanisms of interactions between crop genotypes and species – for example, enhanced resource availability through niche complementarity. Ecological advances include better understanding of the context-dependency of interactions, the mechanisms behind disease and pest avoidance, the links between above- and below-ground systems, and the role of microtopographic variation in coexistence. This improved understanding can guide approaches for improving intercropping systems, including breeding crops for intercropping. Although such advances can help to improve intercropping systems, we suggest that other topics also need addressing. These include better assessment of the wider benefits of intercropping in terms of multiple ecosystem services, collaboration with agricultural engineering, and more effective interdisciplinary research.