Genome-wide association analysis of stalk biomass and anatomical traits in maize.

BACKGROUND: Maize stover is an important source of crop residues and a promising sustainable energy source in the United States. Stalk is the main component of stover, representing about half of stover dry weight. Characterization of genetic determinants of stalk traits provide a foundation to optimize maize stover as a biofuel feedstock. We investigated maize natural genetic variation in genome-wide association studies (GWAS) to detect candidate genes associated with traits related to stalk biomass (stalk diameter and plant height) and stalk anatomy (rind thickness, vascular bundle density and area). RESULTS: Using a panel of 942 diverse inbred lines, 899,784 RNA-Seq derived single nucleotide polymorphism (SNP) markers were identified. Stalk traits were measured on 800 members of the panel in replicated field trials across years. GWAS revealed 16 candidate genes associated with four stalk traits. Most of the detected candidate genes were involved in fundamental cellular functions, such as regulation of gene expression and cell cycle progression. Two of the regulatory genes (Zmm22 and an ortholog of Fpa) that were associated with plant height were previously shown to be involved in regulating the vegetative to floral transition. The association of Zmm22 with plant height was confirmed using a transgenic approach. Transgenic lines with increased expression of Zmm22 showed a significant decrease in plant height as well as tassel branch number, indicating a pleiotropic effect of Zmm22. CONCLUSION: Substantial heritable variation was observed in the association panel for stalk traits, indicating a large potential for improving useful stalk traits in breeding programs. Genome-wide association analyses detected several candidate genes associated with multiple traits, suggesting common regulatory elements underlie various stalk traits. Results of this study provide insights into the genetic control of maize stalk anatomy and biomass.

T-DNA insertion in aquaporin gene AtPIP1;2 generates transcription profiles reminiscent of a low CO2 response.

Results from CO2 diffusion studies and characterization of Arabidopsis thaliana aquaporin AtPIP1;2 T-DNA insertion lines support the idea that specific aquaporins facilitate the diffusion of CO2 through biological membranes. However, their function as CO2 diffusion facilitators in plant physiology is still a matter of debate. Assuming that a lack of AtPIP1;2 causes a characteristic transcriptional response, we compared data from a AtPIP1;2 T-DNA insertion line obtained by Illumina sequencing, Affymetrix chip analysis and quantitative RT-PCR to the transcriptome of plants grown under drought stress or under low CO2 conditions. The plant reaction to the deficit of AtPIP1;2 was unlike drought stress responses but comparable with that of low CO2 conditions. In addition, we observed a phenotype characteristic to plants grown under low CO2 . The findings support the hypothesis that the AtPIP1;2 function in plant physiology is not to facilitate water but CO2 diffusion.

Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize (Zea mays L.) lines.

A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA) content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. This indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment.

The Arabidopsis thaliana aquaporin AtPIP1;2 is a physiologically relevant CO2 transport facilitator.

Cellular exchange of carbon dioxide (CO2) is of extraordinary importance for life. Despite this significance, its molecular mechanisms are still unclear and a matter of controversy. In contrast to other living organisms, plants are physiologically limited by the availability of CO2. In most plants, net photosynthesis is directly dependent on CO2 diffusion from the atmosphere to the chloroplast. Thus, it is important to analyze CO2 transport with regards to its effect on photosynthesis. A mutation of the Arabidopsis thaliana AtPIP1;2 gene, which was characterized as a non-water transporting but CO2 transport-facilitating aquaporin in heterologous expression systems, correlated with a reduction in photosynthesis under a wide range of atmospheric CO2 concentrations. Here, we could demonstrate that the effect was caused by reduced CO2 conductivity in leaf tissue. It is concluded that the AtPIP1;2 gene product limits CO2 diffusion and photosynthesis in leaves.

The Arabidopsis aquaporin PIP1;2 rules cellular CO(2) uptake.

The membrane CO(2) flux into Arabidopsis mesophyll cells was studied using a scanning pH microelectrode. Arabidopsis thaliana mesophyll cells were exposed to photosynthesis-triggering light intensities, which induced cellular CO(2) uptake. Data obtained on a AtPIP1;2 T-DNA insertion line indicated that under these conditions, cellular CO(2) transport was not limited by unstirred layer effects but was dependent on the expression of the aquaporin AtPIP1;2. Complementation of the AtPIP1;2 knockout restored membrane CO(2) transport levels to that of controls. The results provide new arguments for the ongoing debate about the validity of the lipid bilayer model system and the Meyer - Overton rule for cellular gas transport. In conclusion, we suggest a modified model of molecular gas transport mechanisms in living cells.

Aquaporin tetramer composition modifies the function of tobacco aquaporins.

Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO(2)-triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO(2)-related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO(2)-triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO(2)-related transport processes, a structure built up by the tetramer is the basis of this function.

Aquaporins and plant water balance.

The impact of aquaporin function on plant water balance is discussed. The significance of these proteins for root water uptake, water conductance in the xylem, including embolism refilling and the role of plant aquaporins in leaf physiology, is described. Emphasis is placed on certain aspects of water stress reactions and the correlation of aquaporins to abscisic acid as well as on the relation of water and CO2 permeability in leaves.

Suppression of CINNAMOYL-CoA REDUCTASE increases the level of monolignol ferulates incorporated into maize lignins.

BACKGROUND: The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and therefore to the plant body. However, the recalcitrance associated with lignin impedes the extraction of polysaccharides from the cell wall to make plant-based biofuels and biomaterials. The cell wall digestibility can be improved by introducing labile ester bonds into the lignin backbone that can be easily broken under mild base treatment at room temperature. The FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme, which may be naturally found in many plants, uses feruloyl-CoA and monolignols to synthesize the ester-linked monolignol ferulate conjugates. A mutation in the first lignin-specific biosynthetic enzyme, CINNAMOYL-CoA REDUCTASE (CCR), results in an increase in the intracellular pool of feruloyl-CoA. RESULTS: Maize (Zea mays) has a native putative FMT enzyme, and its ccr mutants produce an increased pool of feruloyl-CoA that can be used for conversion to monolignol ferulate conjugates. The decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue. CONCLUSIONS: Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.

Transcriptome analysis of the aquaporin AtPIP1;2 deficient line in Arabidopsis thaliana.

Atmospheric CO2 impacts all aspects of plant development. It has changed in the past and is predicted to change further on. Studies on the response of crop plants to low and elevated CO2 concerning growth, productivity and physiological processes are intense. In contrast, the molecular mechanisms of cellular CO2 exchange are still under discussion. At the same time it becomes more and more accepted that carbon dioxide is transported across cellular biomembranes by CO2 conducting aquaporins. Our recent study (Boudichevskaia et al., 2015) demonstrates that the lack of a single gene product - aquaporin AtPIP1;2 - resulted in massive transcriptional reprogramming in Arabidopsis as a consequence of reduced tissue CO2 diffusion rates. Therefore, the transcriptome data of the aquaporin AtPIP1;2 deficient line can be used in the comparative expression analyses for better understanding the role of aquaporins with regard to CO2 and water transport in plants. Here we describe a gene expression dataset generated for three biological replicates per genotype on Affymetrix platform. We provide detailed methods and analysis on microarray data which has been deposited in Gene Expression Omnibus (GEO): GSE62167. Additionally, we provide the R code for data preprocessing and quality control.

Image analysis of anatomical traits in stalk transections of maize and other grasses.

BACKGROUND: Grass stalks architecturally support leaves and reproductive structures, functionally support the transport of water and nutrients, and are harvested for multiple agricultural uses. Research on these basic and applied aspects of grass stalks would benefit from improved capabilities for measuring internal anatomical features. In particular, methods suitable for phenotyping populations of plants are needed. RESULTS: To meet the need for large-scale measurements of stalk anatomy features, we developed custom image processing software that utilized a variety of global thresholding, local filtering, and feature detection methods to measure rind thickness, pith area, vascular bundle counts, and individual vascular bundle size from digital images of hand-cut transections of stalks collected with a flatbed document scanner. The tool determined vascular bundle number with an average accuracy of 90% across maize genotypes that varied five-fold for this trait. The method is demonstrated on maize, sorghum, and Miscanthus stalks. The computer source code is staged for download. CONCLUSIONS: Simplicity of sample preparation and semi-automated analyses enabled by this tool greatly increase measurement throughput relative to standard microscopy-based techniques while maintaining high accuracy. The tool is expected to be useful in genetic and physiological studies of the relationships between stalk anatomy and traits such as biofuel suitability, water use efficiency, or nutrient transport.