Metabolite and transcript profiling of Guinea grass (Panicum maximum Jacq) response to elevated [CO2] and temperature.

INTRODUCTION: By mid-century, global atmospheric carbon dioxide concentration ([CO2]) is predicted to reach 600 µmol mol-1 with global temperatures rising by 2 °C. Rising [CO2] and temperature will alter the growth and productivity of major food and forage crops across the globe. Although the impact is expected to be greatest in tropical regions, the impact of climate-change has been poorly studied in those regions. OBJECTIVES: This experiment aimed to understand the effects of elevated [CO2] (600 µmol mol-1) and warming (+ 2 °C), singly and in combination, on Panicum maximum Jacq. (Guinea grass) metabolite and transcript profiles. METHODS: We created a de novo assembly of the Panicum maximum transcriptome. Leaf samples were taken at two time points in the Guinea grass growing season to analyze transcriptional and metabolite profiles in plants grown at ambient and elevated [CO2] and temperature, and statistical analyses were used to integrate the data. RESULTS: Elevated temperature altered the content of amino acids and secondary metabolites. The transcriptome of Guinea grass shows a clear time point separations, with the changes in the elevated temperature and [CO2] combination plots. CONCLUSION: Field transcriptomics and metabolomics revealed that elevated temperature and [CO2] result in alterations in transcript and metabolite profiles associated with environmental response, secondary metabolism and stomatal function. These metabolic responses are consistent with greater growth and leaf area production under elevated temperature and [CO2]. These results show that tropical C4 grasslands may have unpredicted responses to global climate change, and that warming during a cool growing season enhances growth and alleviates stress.

High-Throughput Phenotyping of Maize Leaf Physiological and Biochemical Traits Using Hyperspectral Reflectance.

High-throughput, noninvasive field phenotyping has revealed genetic variation in crop morphological, developmental, and agronomic traits, but rapid measurements of the underlying physiological and biochemical traits are needed to fully understand genetic variation in plant-environment interactions. This study tested the application of leaf hyperspectral reflectance (λ = 500-2,400 nm) as a high-throughput phenotyping approach for rapid and accurate assessment of leaf photosynthetic and biochemical traits in maize (Zea mays). Leaf traits were measured with standard wet-laboratory and gas-exchange approaches alongside measurements of leaf reflectance. Partial least-squares regression was used to develop a measure of leaf chlorophyll content, nitrogen content, sucrose content, specific leaf area, maximum rate of phosphoenolpyruvate carboxylation, [CO2]-saturated rate of photosynthesis, and leaf oxygen radical absorbance capacity from leaf reflectance spectra. Partial least-squares regression models accurately predicted five out of seven traits and were more accurate than previously used simple spectral indices for leaf chlorophyll, nitrogen content, and specific leaf area. Correlations among leaf traits and statistical inferences about differences among genotypes and treatments were similar for measured and modeled data. The hyperspectral reflectance approach to phenotyping was dramatically faster than traditional measurements, enabling over 1,000 rows to be phenotyped during midday hours over just 2 to 4 d, and offers a nondestructive method to accurately assess physiological and biochemical trait responses to environmental stress.

Physiological and transcriptomic responses in the seed coat of field-grown soybean (Glycine max L. Merr.) to abiotic stress.

BACKGROUND: Understanding how intensification of abiotic stress due to global climate change affects crop yields is important for continued agricultural productivity. Coupling genomic technologies with physiological crop responses in a dynamic field environment is an effective approach to dissect the mechanisms underpinning crop responses to abiotic stress. Soybean (Glycine max L. Merr. cv. Pioneer 93B15) was grown in natural production environments with projected changes to environmental conditions predicted for the end of the century, including decreased precipitation, increased tropospheric ozone concentrations ([O3]), or increased temperature. RESULTS: All three environmental stresses significantly decreased leaf-level photosynthesis and stomatal conductance, leading to significant losses in seed yield. This was driven by a significant decrease in the number of pods per node for all abiotic stress treatments. To understand the underlying transcriptomic response involved in the yield response to environmental stress, RNA-Sequencing analysis was performed on the soybean seed coat, a tissue that plays an essential role in regulating carbon and nitrogen transport to developing seeds. Gene expression analysis revealed 49, 148 and 1,576 differentially expressed genes in the soybean seed coat in response to drought, elevated [O3] and elevated temperature, respectively. CONCLUSIONS: Elevated [O3] and drought did not elicit substantive transcriptional changes in the soybean seed coat. However, this may be due to the timing of sampling and does not preclude impacts of those stresses on different tissues or different stages in seed coat development. Expression of genes involved in DNA replication and metabolic processes were enriched in the seed coat under high temperate stress, suggesting that the timing of events that are important for cell division and proper seed development were altered in a stressful growth environment.

Elevated ozone reduces photosynthetic carbon gain by accelerating leaf senescence of inbred and hybrid maize in a genotype-specific manner.

Exposure to elevated tropospheric ozone concentration ([O3 ]) accelerates leaf senescence in many C3 crops. However, the effects of elevated [O3 ] on C4 crops including maize (Zea mays L.) are poorly understood in terms of physiological mechanism and genetic variation in sensitivity. Using free air gas concentration enrichment, we investigated the photosynthetic response of 18 diverse maize inbred and hybrid lines to season-long exposure to elevated [O3 ] (~100 nl L-1 ) in the field. Gas exchange was measured on the leaf subtending the ear throughout the grain filling period. On average over the lifetime of the leaf, elevated [O3 ] led to reductions in photosynthetic CO2 assimilation of both inbred (-22%) and hybrid (-33%) genotypes. There was significant variation among both inbred and hybrid lines in the sensitivity of photosynthesis to elevated [O3 ], with some lines showing no change in photosynthesis at elevated [O3 ]. Based on analysis of inbred line B73, the reduced CO2 assimilation at elevated [O3 ] was associated with accelerated senescence decreasing photosynthetic capacity and not altered stomatal limitation. These findings across diverse maize genotypes could advance the development of more O3 tolerant maize and provide experimental data for parameterization and validation of studies modeling how O3 impacts crop performance.

Identification of Multiple Phytotoxins Produced by Fusarium virguliforme Including a Phytotoxic Effector (FvNIS1) Associated With Sudden Death Syndrome Foliar Symptoms.

Sudden death syndrome (SDS) of soybean is caused by a soilborne pathogen, Fusarium virguliforme. Phytotoxins produced by F. virguliforme are translocated from infected roots to leaves, in which they cause SDS foliar symptoms. In this study, additional putative phytotoxins of F. virguliforme were identified, including three secondary metabolites and 11 effectors. While citrinin, fusaric acid, and radicicol induced foliar chlorosis and wilting, Soybean mosaic virus (SMV)-mediated overexpression of F. virguliforme necrosis-inducing secreted protein 1 (FvNIS1) induced SDS foliar symptoms that mimicked the development of foliar symptoms in the field. The expression level of fvnis1 remained steady over time, although foliar symptoms were delayed compared with the expression levels. SMV::FvNIS1 also displayed genotype-specific toxicity to which 75 of 80 soybean cultivars were susceptible. Genome-wide association mapping further identified three single nucleotide polymorphisms at two loci, where three leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes were found. Culture filtrates of fvnis1 knockout mutants displayed a mild reduction in phytotoxicity, indicating that FvNIS1 is one of the phytotoxins responsible for SDS foliar symptoms and may contribute to the quantitative susceptibility of soybean by interacting with the LRR-RLK genes.

Genomic characterization of plant cell wall degrading enzymes and in silico analysis of xylanases and polygalacturonases of Fusarium virguliforme.

BACKGROUND: Plant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliforme was sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme. RESULTS: F. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2). CONCLUSIONS: Identification and annotation of CAZy provided advanced understanding of genomic composition of PCWDEs in F. virguliforme. Sequence and structural analyses of FvXyn11A and FvXyn11B suggested both xylanases were conserved in residues that allow XIP-I inhibition, and expression of both xylanases were detected during soybean roots infection. We postulate that a transgenic soybean expressing wheat XIP-I may be useful for developing root rot resistance to F. virguliforme.

A comparative analysis of transcriptomic, biochemical, and physiological responses to elevated ozone identifies species-specific mechanisms of resilience in legume crops.

Current concentrations of tropospheric ozone ([O3]) pollution negatively impact plant metabolism, which can result in decreased crop yields. Interspecific variation in the physiological response of plants to elevated [O3] exists; however, the underlying cellular responses explaining species-specific differences are largely unknown. Here, a physiological screen has been performed on multiple varieties of legume species. Three varieties of garden pea (Pisum sativum L.) were resilient to elevated [O3]. Garden pea showed no change in photosynthetic capacity or leaf longevity when exposed to elevated [O3], in contrast to varieties of soybean (Glycine max (L.) Merr.) and common bean (Phaseolus vulgaris L.). Global transcriptomic and targeted biochemical analyses were then done to examine the mechanistic differences in legume responses to elevated [O3]. In all three species, there was an O3-mediated reduction in specific leaf weight and total non-structural carbohydrate content, as well as increased abundance of respiration-related transcripts. Differences specific to garden pea included a pronounced increase in the abundance of GLUTATHIONE REDUCTASE transcript, as well as greater contents of foliar glutathione, apoplastic ascorbate, and sucrose in elevated [O3]. These results suggest that garden pea may have had greater capacity for detoxification, which prevented net losses in CO2 fixation in an elevated [O3] environment.

Heat waves imposed during early pod development in soybean (Glycine max) cause significant yield loss despite a rapid recovery from oxidative stress.

Heat waves already have a large impact on crops and are predicted to become more intense and more frequent in the future. In this study, heat waves were imposed on soybean using infrared heating technology in a fully open-air field experiment. Five separate heat waves were applied to field-grown soybean (Glycine max) in central Illinois, three in 2010 and two in 2011. Thirty years of historical weather data from Illinois were analyzed to determine the length and intensity of a regionally realistic heat wave resulting in experimental heat wave treatments during which day and night canopy temperatures were elevated 6 °C above ambient for 3 days. Heat waves were applied during early or late reproductive stages to determine whether and when heat waves had an impact on carbon metabolism and seed yield. By the third day of each heat wave, net photosynthesis (A), specific leaf weight (SLW), and leaf total nonstructural carbohydrate concentration (TNC) were decreased, while leaf oxidative stress was increased. However, A, SLW, TNC, and measures of oxidative stress were no different than the control ca. 12 h after the heat waves ended, indicating rapid physiological recovery from the high-temperature stress. That end of season seed yield was reduced (~10%) only when heat waves were applied during early pod developmental stages indicates the yield loss had more to do with direct impacts of the heat waves on reproductive process than on photosynthesis. Soybean was unable to mitigate yield loss after heat waves given during late reproductive stages. This study shows that short high-temperature stress events that reduce photosynthesis and increase oxidative stress resulted in significant losses to soybean production in the Midwest, U.S. The study also suggests that to mitigate heat wave-induced yield loss, soybean needs improved reproductive and photosynthetic tolerance to high but increasingly common temperatures.

Ozone exposure response for U.S. soybean cultivars: linear reductions in photosynthetic potential, biomass, and yield.

Current background ozone (O(3)) concentrations over the northern hemisphere's midlatitudes are high enough to damage crops and are projected to increase. Soybean (Glycine max) is particularly sensitive to O(3); therefore, establishing an O(3) exposure threshold for damage is critical to understanding the current and future impact of this pollutant. This study aims to determine the exposure response of soybean to elevated tropospheric O(3) by measuring the agronomic, biochemical, and physiological responses of seven soybean genotypes to nine O(3) concentrations (38-120 nL L(-1)) within a fully open-air agricultural field location across 2 years. All genotypes responded similarly, with season-long exposure to O(3) causing a linear increase in antioxidant capacity while reducing leaf area, light absorption, specific leaf mass, primary metabolites, seed yield, and harvest index. Across two seasons with different temperature and rainfall patterns, there was a robust linear yield decrease of 37 to 39 kg ha(-1) per nL L(-1) cumulative O(3) exposure over 40 nL L(-1). The existence of immediate effects of O(3) on photosynthesis, stomatal conductance, and photosynthetic transcript abundance before and after the initiation and termination of O(3) fumigation were concurrently assessed, and there was no evidence to support an instantaneous photosynthetic response. The ability of the soybean canopy to intercept radiation, the efficiency of photosynthesis, and the harvest index were all negatively impacted by O(3), suggesting that there are multiple targets for improving soybean responses to this damaging air pollutant.

Chronic ozone exacerbates the reduction in photosynthesis and acceleration of senescence caused by limited N availability in Nicotiana sylvestris.

Both elevated ozone (O(3)) and limiting soil nitrogen (N) availability negatively affect crop performance. However, less is known about how the combination of elevated O(3) and limiting N affect crop growth and metabolism. In this study, we grew tobacco (Nicotiana sylvestris) in ambient and elevated O(3) at two N levels (limiting and sufficient). Results at the whole plant, leaf, and cellular level showed that primary metabolism was reduced by growth in limiting N, and that reduction was exacerbated by exposure to elevated O(3). Limiting N reduced the rates of photosynthetic CO(2) uptake by 40.8% in ambient O(3)-exposed plants, and by 58.6% in elevated O(3)-exposed plants, compared with plants grown with sufficient N. Reductions in photosynthesis compounded to cause large differences in leaf and whole plant parameters including leaf number, leaf area, and leaf and root biomass. These results were consistent with our meta-analysis of all published studies of plant responses to elevated O(3) and N availability. In tobacco, N uptake and allocation was also affected by growth in limiting N and elevated O(3), and there was an O(3)-induced compensatory response that resulted in increased N recycling from senescing leaves. In addition, transcript-based markers were used to track the progress through senescence, and indicated that limiting N and elevated O(3), separately and in combination, caused an acceleration of senescence. These results suggest that reductions in crop productivity in growing regions with poor soil fertility will be exacerbated by rising background O(3).

Accelerating yield potential in soybean: potential targets for biotechnological improvement.

Soybean (Glycine max Merr.) is the world's most widely grown legume and provides an important source of protein and oil. Global soybean production and yield per hectare increased steadily over the past century with improved agronomy and development of cultivars suited to a wide range of latitudes. In order to meet the needs of a growing world population without unsustainable expansion of the land area devoted to this crop, yield must increase at a faster rate than at present. Here, the historical basis for the yield gains realized in the past 90 years are examined together with potential metabolic targets for achieving further improvements in yield potential. These targets include improving photosynthetic efficiency, optimizing delivery and utilization of carbon, more efficient nitrogen fixation and altering flower initiation and abortion. Optimization of investment in photosynthetic enzymes, bypassing photorespiratory metabolism, engineering the electron transport chain and engineering a faster recovery from the photoprotected state are different strategies to improve photosynthesis in soybean. These potential improvements in photosynthetic carbon gain will need to be matched by increased carbon and nitrogen transport to developing soybean pods and seeds in order to maximize the benefit. Better understanding of control of carbon and nitrogen transport along with improved knowledge of the regulation of flower initiation and abortion will be needed to optimize sink capacity in soybean. Although few single targets are likely to deliver a quantum leap in yields, biotechnological advances in molecular breeding techniques that allow for alteration of the soybean genome and transcriptome promise significant yield gains.

A putative transporter is essential for integrating nutrient and hormone signaling with lateral root growth and nodule development in Medicago truncatula.

Legume root architecture involves not only elaboration of the root system by the formation of lateral roots but also the formation of symbiotic root nodules in association with nitrogen-fixing soil rhizobia. The Medicago truncatula LATD/NIP gene plays an essential role in the development of both primary and lateral roots as well as nodule development. We have cloned the LATD/NIP gene and show that it encodes a member of the NRT1(PTR) transporter family. LATD/NIP is expressed throughout the plant. pLATD/NIP-GFP promoter-reporter fusions in transgenic roots establish the spatial expression of LATD/NIP in primary root, lateral root and nodule meristems and the surrounding cells. Expression of LATD/NIP is regulated by hormones, in particular by abscisic acid which has been previously shown to rescue the primary and lateral root meristem arrest of latd mutants. latd mutants respond normally to ammonium but have defects in responses of the root architecture to nitrate. Taken together, these results suggest that LATD/NIP may encode a nitrate transporter or transporter of another compound.

The bench scientist's guide to statistical analysis of RNA-Seq data.

BACKGROUND: RNA sequencing (RNA-Seq) is emerging as a highly accurate method to quantify transcript abundance. However, analyses of the large data sets obtained by sequencing the entire transcriptome of organisms have generally been performed by bioinformatics specialists. Here we provide a step-by-step guide and outline a strategy using currently available statistical tools that results in a conservative list of differentially expressed genes. We also discuss potential sources of error in RNA-Seq analysis that could alter interpretation of global changes in gene expression. FINDINGS: When comparing statistical tools, the negative binomial distribution-based methods, edgeR and DESeq, respectively identified 11,995 and 11,317 differentially expressed genes from an RNA-seq dataset generated from soybean leaf tissue grown in elevated O3. However, the number of genes in common between these two methods was only 10,535, resulting in 2,242 genes determined to be differentially expressed by only one method. Upon analysis of the non-significant genes, several limitations of these analytic tools were revealed, including evidence for overly stringent parameters for determining statistical significance of differentially expressed genes as well as increased type II error for high abundance transcripts. CONCLUSIONS: Because of the high variability between methods for determining differential expression of RNA-Seq data, we suggest using several bioinformatics tools, as outlined here, to ensure that a conservative list of differentially expressed genes is obtained. We also conclude that despite these analytical limitations, RNA-Seq provides highly accurate transcript abundance quantification that is comparable to qRT-PCR.

The effects of tropospheric ozone on net primary productivity and implications for climate change.

Tropospheric ozone (O(3)) is a global air pollutant that causes billions of dollars in lost plant productivity annually. It is an important anthropogenic greenhouse gas, and as a secondary air pollutant, it is present at high concentrations in rural areas far from industrial sources. It also reduces plant productivity by entering leaves through the stomata, generating other reactive oxygen species and causing oxidative stress, which in turn decreases photosynthesis, plant growth, and biomass accumulation. The deposition of O(3) into vegetation through stomata is an important sink for tropospheric O(3), but this sink is modified by other aspects of environmental change, including rising atmospheric carbon dioxide concentrations, rising temperature, altered precipitation, and nitrogen availability. We review the atmospheric chemistry governing tropospheric O(3) mass balance, the effects of O(3) on stomatal conductance and net primary productivity, and implications for agriculture, carbon sequestration, and climate change.