Constitutively active B2 Raf-like kinases are required for drought-responsive gene expression upstream of ABA-activated SnRK2 kinases.

Osmotic stresses, such as drought and high salinity, adversely affect plant growth and productivity. The phytohormone abscisic acid (ABA) accumulates in response to osmotic stress and enhances stress tolerance in plants by triggering multiple physiological responses through ABA signaling. Subclass III SNF1-related protein kinases 2 (SnRK2s) are key regulators of ABA signaling. Although SnRK2s have long been considered to be self-activated by autophosphorylation after release from PP2C-mediated inhibition, they were recently revealed to be activated by two independent subfamilies of group B Raf-like kinases, B2-RAFs and B3-RAFs, under osmotic stress conditions. However, the relationship between SnRK2 phosphorylation by these RAFs and SnRK2 autophosphorylation and the individual physiological roles of each RAF subfamily remain unknown. In this study, we indicated that B2-RAFs are constantly active and activate SnRK2s when released from PP2C-mediated inhibition by ABA-binding ABA receptors, whereas B3-RAFs are activated only under stress conditions in an ABA-independent manner and enhance SnRK2 activity. Autophosphorylation of subclass III SnRK2s is not sufficient for ABA responses, and B2-RAFs are needed to activate SnRK2s in an ABA-dependent manner. Using plants grown in soil, we found that B2-RAFs regulate subclass III SnRK2s at the early stage of drought stress, whereas B3-RAFs regulate SnRK2s at the later stage. Thus, B2-RAFs are essential kinases for the activation of subclass III SnRK2s in response to ABA under mild osmotic stress conditions, and B3-RAFs function as enhancers of SnRK2 activity under severe stress conditions.

MORE PANICLES 3, a natural allele of OsTB1/FC1, impacts rice yield in paddy fields at elevated CO2 levels.

Improving crop yield potential through an enhanced response to rising atmospheric CO2 levels is an effective strategy for sustainable crop production in the face of climate change. Large-sized panicles (containing many spikelets per panicle) have been a recent ideal plant architecture (IPA) for high-yield rice breeding. However, few breeding programs have proposed an IPA under the projected climate change. Here, we demonstrate through the cloning of the rice (Oryza sativa) quantitative trait locus for MORE PANICLES 3 (MP3) that the improvement in panicle number increases grain yield at elevated atmospheric CO2 levels. MP3 is a natural allele of OsTB1/FC1, previously reported as a negative regulator of tiller bud outgrowth. The temperate japonica allele advanced the developmental process in axillary buds, moderately promoted tillering, and increased the panicle number without negative effects on the panicle size or culm thickness in a high-yielding indica cultivar with large-sized panicles. The MP3 allele, containing three exonic polymorphisms, was observed in most accessions in the temperate japonica subgroups but was rarely observed in the indica subgroup. No selective sweep at MP3 in either the temperate japonica or indica subgroups suggested that MP3 has not been involved and utilized in artificial selection during domestication or breeding. A free-air CO2 enrichment experiment revealed a clear increase of grain yield associated with the temperate japonica allele at elevated atmospheric CO2 levels. Our findings show that the moderately increased panicle number combined with large-sized panicles using MP3 could be a novel IPA and contribute to an increase in rice production under climate change with rising atmospheric CO2 levels.

Transcriptome-based prediction for polygenic traits in rice using different gene subsets.

BACKGROUND: Transcriptome-based prediction of complex phenotypes is a relatively new statistical method that links genetic variation to phenotypic variation. The selection of large-effect genes based on a priori biological knowledge is beneficial for predicting oligogenic traits; however, such a simple gene selection method is not applicable to polygenic traits because causal genes or large-effect loci are often unknown. Here, we used several gene-level features and tested whether it was possible to select a gene subset that resulted in better predictive ability than using all genes for predicting a polygenic trait. RESULTS: Using the phenotypic values of shoot and root traits and transcript abundances in leaves and roots of 57 rice accessions, we evaluated the predictive abilities of the transcriptome-based prediction models. Leaf transcripts predicted shoot phenotypes, such as plant height, more accurately than root transcripts, whereas root transcripts predicted root phenotypes, such as crown root length, more accurately than leaf transcripts. Furthermore, we used the following three features to train the prediction model: (1) tissue specificity of the transcripts, (2) ontology annotations, and (3) co-expression modules for selecting gene subsets. Although models trained by a gene subset often resulted in lower predictive abilities than the model trained by all genes, some gene subsets showed improved predictive ability. For example, using genes expressed in roots but not in leaves, the predictive ability for crown root diameter was improved by more than 10% (R2 = 0.59 when using all genes; R2 = 0.66, using 1,554 root-specifically expressed genes). Similarly, genes annotated as "gibberellic acid sensitivity" showed higher predictive ability than using all genes for root dry weight. CONCLUSIONS: Our results highlight both the possibility and difficulty of selecting an appropriate gene subset to predict polygenic traits from transcript abundance, given the current biological knowledge and information. Further integration of multiple sources of information, as well as improvements in gene characterization, may enable the selection of an optimal gene set for the prediction of polygenic phenotypes.

Life-Cycle Multiomics of Rice Shoots Reveals Growth Stage-Specific Effects of Drought Stress and Time-Lag Drought Responses.

Field-grown rice plants are exposed to various stresses at different stages of their life cycle, but little is known about the effects of stage-specific stresses on phenomes and transcriptomes. In this study, we performed integrated time-course multiomics on rice at 3-d intervals from seedling to heading stage under six drought conditions in a well-controlled growth chamber. Drought stress at seedling and reproductive stages reduced yield performance by reducing seed number and setting rate, respectively. High temporal resolution analysis revealed that drought response occurred in two steps: a rapid response via the abscisic acid (ABA) signaling pathway and a slightly delayed DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN (DREB) pathway, allowing plants to respond flexibly to deteriorating soil water conditions. Our long-term time-course multiomics showed that temporary drought stress delayed flowering due to prolonged expression of the flowering repressor gene GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (Ghd7) and delayed expression of the florigen genes HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Our life-cycle multiomics dataset on rice shoots under drought conditions provides a valuable resource for further functional genomic studies to improve crop resilience to drought stress.

Three-dimensional image analysis specifies the root distribution for drought avoidance in the early growth stage of rice.

BACKGROUND AND AIMS: Root system architecture (RSA) plays a key role in plant adaptation to drought because deep rooting enables better water uptake than shallow rooting under terminal drought. Understanding RSA during early plant development is essential for improving crop yields, as early drought can affect subsequent shoot growth. Herein, we demonstrate that root distribution in the topsoil significantly impacts shoot growth during the early stages of rice (Oryza sativa) development under drought, as assessed through three-dimensional (3D) image analysis. METHODS: We used 109 F12 recombinant inbred lines (RILs) obtained from a cross between shallow-rooting lowland rice and deep-rooting upland rice, representing a population with diverse RSA. We applied a moderate drought during the early development of rice grown in a plant pot (25 cm height) by stopping irrigation 14 days after sowing (DAS). Time-series RSA at 14, 21, and 28 DAS was visualized by X-ray computed tomography, and subsequently compared between drought and well-watered conditions. Following this analysis, we further investigated drought-avoidant RSA by testing 20 randomly selected RILs under drought conditions. KEY RESULTS: We inferred the root location that most influences shoot growth using a hierarchical Bayes approach: the root segment depth, which positively impacted shoot growth, ranged between 1.7-3.4 cm under drought conditions and between 0.0-1.7 cm under well-watered conditions. Drought-avoidant RILs had a higher root density in the lower layers of the topsoil compared to the others. CONCLUSIONS: Fine classification of soil layers using 3D image analysis revealed that increasing root density in the lower layers of the topsoil, rather than in the subsoil, is advantageous for drought avoidance during the early growth stage of rice.

Root angle modifications by the DRO1 homolog improve rice yields in saline paddy fields.

The root system architecture (RSA) of crops can affect their production, particularly in abiotic stress conditions, such as with drought, waterlogging, and salinity. Salinity is a growing problem worldwide that negatively impacts on crop productivity, and it is believed that yields could be improved if RSAs that enabled plants to avoid saline conditions were identified. Here, we have demonstrated, through the cloning and characterization of qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1), that a shallower root growth angle (RGA) could enhance rice yields in saline paddies. qSOR1 is negatively regulated by auxin, predominantly expressed in root columella cells, and involved in the gravitropic responses of roots. qSOR1 was found to be a homolog of DRO1 (DEEPER ROOTING 1), which is known to control RGA. CRISPR-Cas9 assays revealed that other DRO1 homologs were also involved in RGA. Introgression lines with combinations of gain-of-function and loss-of-function alleles in qSOR1 and DRO1 demonstrated four different RSAs (ultra-shallow, shallow, intermediate, and deep rooting), suggesting that natural alleles of the DRO1 homologs could be utilized to control RSA variations in rice. In saline paddies, near-isogenic lines carrying the qSOR1 loss-of-function allele had soil-surface roots (SOR) that enabled rice to avoid the reducing stresses of saline soils, resulting in increased yields compared to the parental cultivars without SOR. Our findings suggest that DRO1 homologs are valuable targets for RSA breeding and could lead to improved rice production in environments characterized by abiotic stress.

Development of 12 sets of chromosome segment substitution lines that enhance allele mining in Asian cultivated rice.

Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.

The transcriptomic landscapes of rice cultivars with diverse root system architectures grown in upland field conditions.

Root system architecture affects plant drought resistance and other key agronomic traits such as lodging. However, although phenotypic and genomic variation has been extensively analyzed, few field studies have integrated phenotypic and transcriptomic information, particularly for below-ground traits such as root system architecture. Here, we report the phenotypic and transcriptomic landscape of 61 rice (Oryza sativa) accessions with highly diverse below-ground traits grown in an upland field. We found that four principal components explained the phenotypic variation and that accessions could be classified into four subpopulations (indica, aus, japonica and admixed) based on their tiller numbers and crown root diameters. Transcriptome analysis revealed that differentially expressed genes associated with specific subpopulations were enriched with stress response-related genes, suggesting that subpopulations have distinct stress response mechanisms. Root growth was negatively correlated with auxin-inducible genes, suggesting an association between auxin signaling and upland field conditions. A negative correlation between crown root diameter and stress response-related genes suggested that thicker crown root diameter is associated with resistance to mild drought stress. Finally, co-expression network analysis implemented with DNA affinity purification followed by sequencing analysis identified phytohormone signaling networks and key transcription factors negatively regulating crown root diameter. Our datasets provide a useful resource for understanding the genomic and transcriptomic basis of phenotypic variation under upland field conditions.

iPOTs: Internet of Things-based pot system controlling optional treatment of soil water condition for plant phenotyping under drought stress.

A cultivation facility that can assist users in controlling the soil water condition is needed for accurately phenotyping plants under drought stress in an artificial environment. Here we report the Internet of Things-based pot system controlling optional treatment of soil water condition (iPOTs), an automatic irrigation system that mimics the drought condition in a growth chamber. The Wi-Fi-enabled iPOTs system allows water supply from the bottom of the pot, based on the soil water level set by the user, and automatically controls the soil water level at a desired depth. The iPOTs also allows users to monitor environmental parameters, such as soil temperature, air temperature, humidity, and light intensity, in each pot. To verify whether the iPOTs mimics the drought condition, we conducted a drought stress test on rice (Oryza sativa L.) varieties and near-isogenic lines, with diverse root system architecture, using the iPOTs system installed in a growth chamber. Similar to the results of a previous drought stress field trial, the growth of shallow-rooted rice accessions was severely affected by drought stress compared with that of deep-rooted accessions. The microclimate data obtained using the iPOTs system increased the accuracy of plant growth evaluation. Transcriptome analysis revealed that pot positions in the growth chamber had little impact on plant growth. Together, these results suggest that the iPOTs system is a reliable platform for phenotyping plants under drought stress.

Improving the efficiency of plant root system phenotyping through digitization and automation.

Root system architecture (RSA) determines unevenly distributed water and nutrient availability in soil. Genetic improvement of RSA, therefore, is related to crop production. However, RSA phenotyping has been carried out less frequently than above-ground phenotyping because measuring roots in the soil is difficult and labor intensive. Recent advancements have led to the digitalization of plant measurements; this digital phenotyping has been widely used for measurements of both above-ground and RSA traits. Digital phenotyping for RSA is slower and more difficult than for above-ground traits because the roots are hidden underground. In this review, we summarized recent trends in digital phenotyping for RSA traits. We classified the sample types into three categories: soil block containing roots, section of soil block, and root sample. Examples of the use of digital phenotyping are presented for each category. We also discussed room for improvement in digital phenotyping in each category.

Identification of a unique allele in the quantitative trait locus for crown root number in japonica rice from Japan using genome-wide association studies.

To explore the genetic resources that could be utilized to help improve root system architecture phenotypes in rice (Oryza sativa), we have conducted genome-wide association studies to investigate maximum root length and crown root number in 135 10-day-old Japanese rice accessions grown hydroponically. We identified a quantitative trait locus for crown root number at approximately 32.7 Mbp on chromosome 4 and designated it qNCR1 (quantitative trait locus for Number of Crown Root 1). A linkage disequilibrium map around qNCR1 suggested that three candidate genes are involved in crown root number: a cullin (LOC_Os04g55030), a gibberellin 20 oxidase 8 (LOC_Os04g55070), and a cyclic nucleotide-gated ion channel (LOC_Os04g55080). The combination of haplotypes for each gene was designated as a haploblock, and haploblocks 1, 2, and 3 were defined. Compared to haploblock 1, the accessions with haploblocks 2 and 3 had fewer crown roots; approximately 5% and 10% reductions in 10-day-old plants and 15% and 25% reductions in 42-day-old plants, respectively. A Japanese leading variety Koshihikari and its progenies harbored haploblock 3. Their crown root number could potentially be improved using haploblocks 1 and 2.

RSAtrace3D: robust vectorization software for measuring monocot root system architecture.

BACKGROUND: The root distribution in the soil is one of the elements that comprise the root system architecture (RSA). In monocots, RSA comprises radicle and crown roots, each of which can be basically represented by a single curve with lateral root branches or approximated using a polyline. Moreover, RSA vectorization (polyline conversion) is useful for RSA phenotyping. However, a robust software that can enable RSA vectorization while using noisy three-dimensional (3D) volumes is unavailable. RESULTS: We developed RSAtrace3D, which is a robust 3D RSA vectorization software for monocot RSA phenotyping. It manages the single root (radicle or crown root) as a polyline (a vector), and the set of the polylines represents the entire RSA. RSAtrace3D vectorizes root segments between the two ends of a single root. By utilizing several base points on the root, RSAtrace3D suits noisy images if it is difficult to vectorize it using only two end nodes of the root. Additionally, by employing a simple tracking algorithm that uses the center of gravity (COG) of the root voxels to determine the tracking direction, RSAtrace3D efficiently vectorizes the roots. Thus, RSAtrace3D represents the single root shape more precisely than straight lines or spline curves. As a case study, rice (Oryza sativa) RSA was vectorized from X-ray computed tomography (CT) images, and RSA traits were calculated. In addition, varietal differences in RSA traits were observed. The vector data were 32,000 times more compact than raw X-ray CT images. Therefore, this makes it easier to share data and perform re-analyses. For example, using data from previously conducted studies. For monocot plants, the vectorization and phenotyping algorithm are extendable and suitable for numerous applications. CONCLUSIONS: RSAtrace3D is an RSA vectorization software for 3D RSA phenotyping for monocots. Owing to the high expandability of the RSA vectorization and phenotyping algorithm, RSAtrace3D can be applied not only to rice in X-ray CT images but also to other monocots in various 3D images. Since this software is written in Python language, it can be easily modified and will be extensively applied by researchers in this field.

Challenges to design-oriented breeding of root system architecture adapted to climate change.

Roots are essential organs for capturing water and nutrients from the soil. In particular, root system architecture (RSA) determines the extent of the region of the soil where water and nutrients can be gathered. As global climate change accelerates, it will be important to improve belowground plant parts, as well as aboveground ones, because roots are front-line organs in the response to abiotic stresses such as drought, flooding, and salinity stress. However, using conventional breeding based on phenotypic selection, it is difficult to select breeding lines possessing promising RSAs to adapted to abiotic stress because roots remain hidden underground. Therefore, new breeding strategies that do not require phenotypic selection are necessary. Recent advances in molecular biology and biotechnology can be applied to the design-oriented breeding of RSA without phenotypic selection. Here I summarize recent progress in RSA ideotypes as "design" and RSA-related gene resources as "materials" that will be needed in leveraging these technologies for the RSA breeding. I also highlight the future challenges to design-oriented breeding of RSA and explore solutions to these challenges.

The intersection of nitrogen nutrition and water use in plants: new paths toward improved crop productivity.

Water and nitrogen availability limit crop productivity globally more than most other environmental factors. Plant availability of macronutrients such as nitrate is, to a large extent, regulated by the amount of water available in the soil, and, during drought episodes, crops can become simultaneously water and nitrogen limited. In this review, we explore the intricate relationship between water and nitrogen transport in plants, from transpiration-driven mass flow in the soil to uptake by roots via membrane transporters and channels and transport to aerial organs. We discuss the roles of root architecture and of suberized hydrophobic root barriers governing apoplastic water and nitrogen movement into the vascular system. We also highlight the need to identify the signalling cascades regulating water and nitrogen transport, as well as the need for targeted physiological analyses of plant traits influencing water and nitrogen uptake. We further advocate for incorporation of new phenotyping technologies, breeding strategies, and agronomic practices to improve crop yield in water- and nitrogen-limited production systems.

Backhoe-assisted monolith method for plant root phenotyping under upland conditions.

Root system architecture (RSA) is one of the most important traits determining water and nutrient availability for plants. Modification of RSA is known to be a useful approach for improving root performance of crops. However, for conducting root phenotyping, there are few alternatives for the rapid collection of root samples from a constant soil volume. In this report, we propose a rapid root-sampling method, which uses a steel cylinder known as round monolith and backhoes to reduce the physical effort. The monolith was set on the ground surrounding individual rice plants and vertically driven back by a backhoe. Soil samples with 20 cm width and 25 cm depth were excavated by the monolith, from which root samples were then isolated. This backhoe-assisted monolith method requires at most five minutes to collect root samples from one plant. Using this method, we quantified the root traits of three rice lines, reported to form different types of root system such as shallow-, intermediate-, and deep-roots, using a root image analysis software. The data obtained through this method, which showed the same trend as previously reported, clearly demonstrated that this method is useful for quantitative evaluation of roots in the soil.

Genomic regions responsible for seminal and crown root lengths identified by 2D & 3D root system image analysis.

BACKGROUND: Genetic improvement of root system architecture is a promising approach for improved uptake of water and mineral nutrients distributed unevenly in the soil. To identify genomic regions associated with the length of different root types in rice, we quantified root system architecture in a set of 26 chromosome segment substitution lines derived from a cross between lowland indica rice, IR64, and upland tropical japonica rice, Kinandang Patong, (IK-CSSLs), using 2D & 3D root phenotyping platforms. RESULTS: Lengths of seminal and crown roots in the IK-CSSLs grown under hydroponic conditions were measured by 2D image analysis (RootReader2D). Twelve CSSLs showed significantly longer seminal root length than the recurrent parent IR64. Of these, 8 CSSLs also exhibited longer total length of the three longest crown roots compared to IR64. Three-dimensional image analysis (RootReader3D) for these CSSLs grown in gellan gum revealed that only one CSSL, SL1003, showed significantly longer total root length than IR64. To characterize the root morphology of SL1003 under soil conditions, SL1003 was grown in Turface, a soil-like growth media, and roots were quantified using RootReader3D. SL1003 had larger total root length and increased total crown root length than did IR64, although its seminal root length was similar to that of IR64. The larger TRL in SL1003 may be due to increased crown root length. CONCLUSIONS: SL1003 carries an introgression from Kinandang Patong on the long arm of chromosome 1 in the genetic background of IR64. We conclude that this region harbors a QTL controlling crown root elongation.

Genetic variation of root angle distribution in rice (Oryza sativa L.) seedlings.

We developed a new method of using seedling trays to evaluate root angle distribution in rice (Oryza sativa. L), and found a wide genetic variation among cultivars. The seedling tray method can be used to evaluate in detail the growth angles of rice crown roots at the seedling stage by allocating nine scores (10° to 90°). Unlike basket methods, it can handle large plant populations over a short growth period (only 14 days). By using the method, we characterized the root angle distributions of 97 accessions into two cluster groups: A and B. The numbers of accessions in group A were limited, and these were categorized as shallow rooting types including soil-surface root. Group B included from shallow to deep rooting types; both included Indica and Japonica Group cultivars, lowland and upland cultivars, and landraces and improved types. No relationship between variation in root vertical angle and total root number was found. The variation in root angle distribution was not related to differentiation between the Japonica and Indica Groups, among ecosystems used for rice cultivation, or among degrees of genetic improvement. The new evaluation method and associated information on genetic variation of rice accessions will be useful in root architecture breeding of rice.

Genetic architecture of variation in heading date among Asian rice accessions.

BACKGROUND: Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions. RESULTS: To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes. CONCLUSIONS: Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice.

Genetic improvement for root growth angle to enhance crop production.

The root system is an essential organ for taking up water and nutrients and anchoring shoots to the ground. On the other hand, the root system has rarely been regarded as breeding target, possibly because it is more laborious and time-consuming to evaluate roots (which require excavation) in a large number of plants than aboveground tissues. The root growth angle (RGA), which determines the direction of root elongation in the soil, affects the area in which roots capture water and nutrients. In this review, we describe the significance of RGA as a potential trait to improve crop production, and the physiological and molecular mechanisms that regulate RGA. We discuss the prospects for breeding to improve RGA based on current knowledge of quantitative trait loci for RGA in rice.