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High-throughput root phenotyping in the soil became an indispensable quantitative tool for the assessment of effects of climatic factors and molecular perturbation on plant root morphology, development and function. To efficiently analyse a large amount of structurally complex soil-root images advanced methods for automated image segmentation are required. Due to often unavoidable overlap between the intensity of fore- and background regions simple thresholding methods are, generally, not suitable for the segmentation of root regions. Higher-level cognitive models such as convolutional neural networks (CNN) provide capabilities for segmenting roots from heterogeneous and noisy background structures, however, they require a representative set of manually segmented (ground truth) images. Here, we present a GUI-based tool for fully automated quantitative analysis of root images using a pre-trained CNN model, which relies on an extension of the U-Net architecture. The developed CNN framework was designed to efficiently segment root structures of different size, shape and optical contrast using low budget hardware systems. The CNN model was trained on a set of 6465 masks derived from 182 manually segmented near-infrared (NIR) maize root images. Our experimental results show that the proposed approach achieves a Dice coefficient of 0.87 and outperforms existing tools (e.g., SegRoot) with Dice coefficient of 0.67 by application not only to NIR but also to other imaging modalities and plant species such as barley and arabidopsis soil-root images from LED-rhizotron and UV imaging systems, respectively. In summary, the developed software framework enables users to efficiently analyse soil-root images in an automated manner (i.e. without manual interaction with data and/or parameter tuning) providing quantitative plant scientists with a powerful analytical tool.

Quantitative characterization of root system architecture and its development is important for the assessment of a complete plant phenotype. To enable high-throughput phenotyping of plant roots efficient solutions for automated image analysis are required. Since plants naturally grow in an opaque soil environment, automated analysis of optically heterogeneous and noisy soil-root images represents a challenging task. Here, we present a user-friendly GUI-based tool for semi-automated analysis of soil-root images which allows to perform an efficient image segmentation using a combination of adaptive thresholding and morphological filtering and to derive various quantitative descriptors of the root system architecture including total length, local width, projection area, volume, spatial distribution and orientation. The results of our semi-automated root image segmentation are in good conformity with the reference ground-truth data (mean dice coefficient = 0.82) compared to IJ_Rhizo and GiAroots. Root biomass values calculated with our tool within a few seconds show a high correlation (Pearson coefficient = 0.8) with the results obtained using conventional, pure manual segmentation approaches. Equipped with a number of adjustable parameters and optional correction tools our software is capable of significantly accelerating quantitative analysis and phenotyping of soil-, agar- and washed root images.

In recent years, various automated methods for plant phenotyping addressing roots or shoots have been developed and corresponding platforms have been established to meet the diverse requirements of plant research and breeding. However, most platforms are only either able to phenotype shoots or roots of plants but not both simultaneously. This substantially limits the opportunities offered by a joint assessment of the growth and development dynamics of both organ systems, which are highly interdependent. In order to overcome these limitations, a root phenotyping installation was integrated into an existing automated non-invasive high-throughput shoot phenotyping platform. Thus, the amended platform is now capable of conducting high-throughput phenotyping at the whole-plant level, and it was used to assess the vegetative root and shoot growth dynamics of five maize inbred lines and four hybrids thereof, as well as the responses of five inbred lines to progressive drought stress. The results showed that hybrid vigour (heterosis) occurred simultaneously in roots and shoots and was detectable as early as 4 days after transplanting (4 DAT; i.e., 8 days after seed imbibition) for estimated plant height (EPH), total root length (TRL), and total root volume (TRV). On the other hand, growth dynamics responses to progressive drought were different in roots and shoots. While TRV was significantly reduced 10 days after the onset of the water deficit treatment, the estimated shoot biovolume was significantly reduced about 6 days later, and EPH showed a significant decrease even 2 days later (8 days later than TRV) compared with the control treatment. In contrast to TRV, TRL initially increased in the water deficit period and decreased much later (not earlier than 16 days after the start of the water deficit treatment) compared with the well-watered plants. This may indicate an initial response of the plants to water deficit by forming longer but thinner roots before growth was inhibited by the overall water deficit. The magnitude and the dynamics of the responses were genotype-dependent, as well as under the influence of the water consumption, which was related to plant size.

Inflorescence architecture of barley (Hordeum vulgare L.) is common among the Triticeae species, which bear one to three singleflowered spikelets at each rachis internode. Triple spikelet meristem is one of the unique features of barley spikes, in which three spikelets (one central and two lateral spikelets) are produced at each rachis internode. Fertility of the lateral spikelets at triple spikelet meristem gives row-type identity to barley spikes. Sixrowed spikes show fertile lateral spikelets and produce increased grain yield per spike, compared with two-rowed spikes with sterile lateral spikelets. Thus, far, two loci governing the row-type phenotype were isolated in barley that include Six-rowed spikei (Vrs1) and Intermedium-C. In the present study, we isolated Six-rowed spike4 (Vrs4), a barley ortholog of the maize (Zea mays L.) inflorescence architecture gene RAMOSA2 (RA2). Eighteen coding mutations in barley RA2 (HvRA2) were specifically associated with lateral spikelet fertility and loss of spikelet determinacy. Expression analyses through mRNA in situ hybridization and microarray showed that Vrs4 (HvRA2) controls the row-type pathway through Vrs1 (HvHox1) a negative regulator of lateral spikelet fertility in barley. Moreover, Vrs4 may also regulate transcripts of barley SISTER OF RAMOSA3 (HvSRA), a putative trehalose-6-phosphate phosphatase involved in trehalose-6-phosphate homeostasis implicated to control spikelet determinacy. Our expression data illustrated that, although RA2 is conserved among different grass species, its down-stream target genes appear to be modified in barley and possibly other species of tribe Triticeae.

Crop plants are regularly challenged by a range of environmental stresses which typically retard their growth and ultimately compromise economic yield. The stress response involves the reprogramming of approximately 4% of the transcriptome. Here, the behavior of AtRD22 and AtUSPL1, both members of the Arabidopsis thaliana BURP (BNM2, USP, RD22 and polygalacturonase isozyme) domain-containing gene family, has been characterized. Both genes are up-regulated as part of the abscisic acid (ABA) mediated moisture stress response. While AtRD22 transcript was largely restricted to the leaf, that of AtUSPL1 was more prevalent in the root. As the loss of function of either gene increased the plant's moisture stress tolerance, the implication was that their products act to suppress the drought stress response. In addition to the known involvement of AtUSPL1 in seed development, a further role in stress tolerance was demonstrated. Based on transcriptomic data and phenotype we concluded that the enhanced moisture stress tolerance of the two loss-of-function mutants is a consequence of an enhanced basal defense response.

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp- 17.5-CI protein suggests that the a-crystallin domain is evolutionarily highly conserved

Rice lines with slower starch digestibility provide opportunities in mitigating the global rise in type II diabetes and related non-communicable diseases. However, screening for low glycemic index (GI) in rice breeding programs is not possible due to time and cost constraints. This study evaluated the feasibility of using in vitro cooked grain amylolysis, starch mobilization patterns during seed germination, and variation in starch structure and composition in the mature seed to differentiate patterns of starch digestibility. Mobilization patterns of total starch, resistant starch, amylose and amylopectin chains, and free sugars during seed germination revealed that the process is analogous to digestion in the human gastrointestinal tract. The combination of these biochemical markers can be used as an alternative measure to predict GI. Additionally, transcriptome analysis of stored mRNA transcripts in high and low GI lines detected differences in starch metabolism and confirmed the importance of seed storage pathways in influencing digestibility. Pathway analyses supported by metabolomics data revealed that resistant starch, cell wall non-starch polysaccharides and flavonoids potentially contribute to slower digestibility. These new insights can guide precision breeding programs to produce low GI rice with acceptable cooking quality to help mitigate the burden of diet-associated lifestyle diseases.

Background Barley ( Hordeum vulgare ) is an important crop cultivated across the world. Drought is a major abiotic factor compromising barley yield worldwide, therefore in modern spring barley cultivars superior seed and malting quality characteristics should be combined with reasonable level of drought tolerance. Previously we have identified a number of barley lines demonstrating the superior yield performance under drought conditions. The aim of this work was to perform a QTL analysis of malting quality traits in a doubled haploid (DH) mapping population of two elite barley lines that differ in their reaction pattern to drought stress. Results A population of DH lines was developed by crossing two drought-tolerant elite breeding lines, Victoriana and Sofiara, exploiting distinct mechanism of drought tolerance, sustaining assimilation vs remobilization. The mapping population was assayed under field conditions at four distinct locations that differed in precipitation rate. DH lines were genotyped with the Illumina 9 K iSelect assay, and linkage map including 1782 polymorphic markers and covering a total map length of 1140 cM was constructed. The result of quantitative trait loci (QTL) analysis showed that majority of the traits were affected by several main effect QTL and/or QTL x environment (QE) interactions. In total, 57, 41, and 5 QTL were associated with yield-related traits, malting quality traits and seed quality traits, respectively. 11 and 29 of mapped QTL explained more than 10 and 5% of phenotypic variation, respectively. In several chromosomal regions co-localization between QTL for various traits were observed. The largest clusters were detected on chromosomes 3H and 4H. Conclusions Our QTL mapping results revealed several novel consistent genomic regions controlling malting quality which could be exploited in marker assisted selection. In this context, the complex QTL region on chromosome 3H seems of particular interest, as it harbors several large effect QTL.

Background Sucrose phosphate synthase (SPS) is an important component of the plant sucrose biosynthesis pathway. In the monocotyledonous Poaceae, five SPS genes have been identified. Here we present a detailed analysis of the wheat SPSII family in wheat. A set of homoeologue-specific primers was developed in order to permit both the detection of sequence variation, and the dissection of the individual contribution of each homoeologue to the global expression of SPSII . Results The expression in bread wheat over the course of development of various sucrose biosynthesis genes monitored on an Affymetrix array showed that the SPS genes were regulated over time and space. SPSII homoeologue-specific assays were used to show that the three homoeologues contributed differentially to the global expression of SPSII . Genetic mapping placed the set of homoeoloci on the short arms of the homoeologous group 3 chromosomes. A resequencing of the A and B genome copies allowed the detection of four haplotypes at each locus. The 3B copy includes an unspliced intron. A comparison of the sequences of the wheat SPSII orthologues present in the diploid progenitors einkorn, goatgrass and Triticum speltoides , as well as in the more distantly related species barley, rice, sorghum and purple false brome demonstrated that intronic sequence was less well conserved than exonic. Comparative sequence and phylogenetic analysis of SPSII gene showed that false purple brome was more similar to Triticeae than to rice. Wheat - rice synteny was found to be perturbed at the SPS region. Conclusion The homoeologue-specific assays will be suitable to derive associations between SPS functionality and key phenotypic traits. The amplicon sequences derived from the homoeologue-specific primers are informative regarding the evolution of SPSII in a polyploid context.

Drought is one of the most severe environmental stress factors limiting crop yield especially when occurring during anthesis and seed filling. This terminal drought is characterized by an excess production of the phytohormone abscisic acid (ABA) which plays an important role during seed development and dormancy. All the genes putatively involved in ABA biosynthesis and inactivation in barley were identified and their expression studied during plant ontogeny under standard and drought-stress conditions to learn more about ABA homeostasis and the possible mode of cross-talk between source and sink tissues. Out of 41 genes related to ABA biosynthesis and inactivation 19 were found to be differentially regulated under drought stress in both flag leaves and developing seed during seed filling. Transcripts of plastid-located enzymes are regulated similarly in flag leaf and seed under terminal drought whereas transcripts of cytosolic enzymes are differentially regulated in the two tissues. Detailed information on the expression of defined gene family members is supplemented by measurements of ABA and its degradation and conjugation products, respectively. Under drought stress, flag leaves in particular contain high concentrations of both ABA and the ABA degradation products phaseic acid (PA) and diphaseic acid (DPA); whereas, in seeds, besides ABA, DPA was mainly found. The measurements also revealed a positive correlation between ABA level and starch content in developing seeds for the following reasons: (i) genes of the ABA controlled SnRK2.6 and RCAR/PP2C-mediated signal transduction pathway to the ABF transcription factor HvABI5 are activated in the developing grain under drought, (ii) novel ABA- and dehydration-responsive cis-elements have been found in the promoters of key genes of starch biosynthesis (HvSUS1, HvAGP-L1) and degradation (HvBAM1) and these transcripts/activity are prominently induced in developing seeds during 12 and 16 DAF, (iii) spraying of fluridone (an ABA biosynthesis inhibitor) to drought-stressed plants results in severely impaired starch content and thousand grain weight of mature seeds.