Stability of grain zinc concentrations across lowland rice environments favors zinc biofortification breeding.

Introduction: One-third of the human population consumes insufficient zinc (Zn) to sustain a healthy life. Zn deficiency can be relieved by increasing the Zn concentration ([Zn]) in staple food crops through biofortification breeding. Rice is a poor source of Zn, and in countries predominantly relying on rice without sufficient dietary diversification, such as Madagascar, Zn biofortification is a priority. Methods: Multi-environmental trials were performed in Madagascar over two years, 2019 and 2020, to screen a total of 28 genotypes including local and imported germplasm. The trials were conducted in the highlands of Ankazomiriotra, Anjiro, and Behenji and in Morovoay, a location representative of the coastal ecosystem. Contributions of genotype (G), environment (E), and G by E interactions (GEIs) were investigated. Result: The grain [Zn] of local Malagasy rice varieties was similar to the internationally established grain [Zn] baseline of 18-20 µg/g for brown rice. While several imported breeding lines reached 50% of our breeding target set at +12 µg/g, only few met farmers' appreciation criteria. Levels of grain [Zn] were stable across E. The G effects accounted for a main fraction of the variation, 76% to 83% of the variation for year 1 and year 2 trials, respectively, while GEI effects were comparatively small, contributing 23% to 9%. This contrasted with dominant E and GEI effects for grain yield. Our results indicate that local varieties tested contained insufficient Zn to alleviate Zn malnutrition, and developing new Zn-biofortified varieties should therefore be a priority. GGE analysis did not distinguish mega-environments for grain [Zn], whereas at least three mega-environments existed for grain yield, differentiated by the presence of limiting environmental conditions and responsiveness to improved soil fertility. Discussion: Our main conclusion reveals that grain [Zn] seems to be under strong genetic control in the agro-climatic conditions of Madagascar. We could identify several interesting genotypes as potential donors for the breeding program, among those BF156, with a relatively stable grain [Zn] (AMMI stability value (ASV) = 0.89) reaching our target (>26 µg/g). While selection for grain yield, general adaptation, and farmers' appreciation would have to rely on multi-environment testing, selection for grain [Zn] could be centralized in earlier generations.

Novel QTL for Lateral Root Density and Length Improve Phosphorus Uptake in Rice (Oryza sativa L.).

The rice root system consists of two types of lateral roots, indeterminate larger L-types capable of further branching, and determinate, short, unbranched S-types. L-type laterals correspond to the typical lateral roots of cereals whereas S-type laterals are unique to rice. Both types contribute to nutrient and water uptake and genotypic variation for density and length of these laterals could be exploited in rice improvement to enhance adaptations to nutrient and water-limited environments. Our objectives were to determine how best to screen for lateral root density and length and to identify markers linked to genotypic variation for these traits. Using different growing media showed that screening in nutrient solution exposed genotypic variation for S-type and L-type density, but only the lateral roots of soil-grown plants varied for their lengths. A QTL mapping population developed from parents contrasting for lateral root traits was grown in a low-P field, roots were sampled, scanned and density and length of lateral roots measured. One QTL each was detected for L-type density (LDC), S-type density on crown root (SDC), S-type density on L-type (SDL), S-type length on L-type (SLL), and crown root number (RNO). The QTL for LDC on chromosome 5 had a major effect, accounting for 46% of the phenotypic variation. This strong positive effect was confirmed in additional field experiments, showing that lines with the donor parent allele at qLDC5 had 50% higher LDC. Investigating the contribution of lateral root traits to P uptake using stepwise regressions indicated LDC and RNO were most influential, followed by SDL. Simulating effects of root trait differences conferred by the main QTL in a P uptake model confirmed that qLDC5 was most effective in improving P uptake followed by qRNO9 for RNO and qSDL9 for S-type lateral density on L-type laterals. Pyramiding qLDC5 with qRNO9 and qSDL9 would be possible given that trade-offs between traits were not detected. Phenotypic selection for the RNO trait during variety development would be feasible, however, the costs of doing so reliably for lateral root density traits is prohibitive and markers identified here therefore provide the first opportunity to incorporate such traits into a breeding program.

Magnesium supply alleviates iron toxicity-induced leaf bronzing in rice through exclusion and tissue-tolerance mechanisms.

Introduction: Iron (Fe) toxicity is a widespread nutritional disorder in lowland rice causing growth retardation and leaf symptoms referred to as leaf bronzing. It is partly caused by an imbalance of nutrients other than Fe and supply of these is known to mitigate the toxicity. But the physiological and molecular mechanisms involved are unknown. Methods: We investigated the effect of magnesium (Mg) on Fe toxicity tolerance in a field study in the Central Highlands of Madagascar and in hydroponic experiments with excess Fe (300 mg Fe L-1). An RNA-seq analysis was conducted in a hydroponic experiment to elucidate possible mechanisms underlying Mg effects. Results and discussion: Addition of Mg consistently decreased leaf bronzing under both field and hydroponic conditions, whereas potassium (K) addition caused minor effects. Plants treated with Mg tended to have smaller shoot Fe concentrations in the field, suggesting enhanced exclusion at the whole-plant level. However, analysis of multiple genotypes showed that Fe toxicity symptoms were also mitigated without a concomitant decrease of Fe concentration, suggesting that increased Mg supply confers tolerance at the tissue level. The hydroponic experiments also suggested that Mg mitigated leaf bronzing without significantly decreasing Fe concentration or oxidative stress as assessed by the content of malondialdehyde, a biomarker for oxidative stress. An RNA-seq analysis revealed that Mg induced more changes in leaves than roots. Subsequent cis-element analysis suggested that NAC transcription factor binding sites were enriched in genes induced by Fe toxicity in leaves. Addition of Mg caused non-significant enrichment of the same binding sites, suggesting that NAC family proteins may mediate the effect of Mg. This study provides clues for mitigating Fe toxicity-induced leaf bronzing in rice.

QTL mapping for early root and shoot vigor of upland rice (Oryza sativa L.) under P deficient field conditions in Japan and Madagascar.

Upland rice production is limited by the low phosphorus (P) availability of many highly weathered tropical soils and P deficiency is likely to become increasingly limiting in future drier climates because P mobility decreases sharply with soil moisture. Good seedling root development will be crucial to cope with the combined effects of low P and water availability. Upland rice genebank accession DJ123 was used as a donor for P efficiency and root vigor traits in a cross with inefficient local variety Nerica4 and a set of backcross lines were used to characterize the seedling stage response of upland rice to low P availability and to identify associated QTL in field trials in Japan and Madagascar. Ten QTL were detected for crown root number, root, shoot and total dry weight per plant in a highly P deficient field in Japan using the BC1F3 generation. Of these, qPef9 on chromosome 9 affected multiple traits, increasing root number, root weight and total biomass, whereas a neighboring QTL on chromosome 9 (qPef9-2) increased shoot biomass. Field trials with derived BC1F5 lines in a low-P field in Madagascar confirmed a highly influential region on chromosome 9. However, qPef9-2 appeared more influential than qPef9, as the shoot and root biomass contrast between lines carrying DJ123 or Nerica4 alleles at qPef9-2 was +23.8% and +13.5% compared to +19.2% and +14.4% at qPef9. This advantage increased further during the growing season, leading to 46% higher shoot biomass at the late vegetative stage. Results suggest an introgression between 8.0 and 12.9 Mb on chromosome 9 from P efficient donor DJ123 can improve plant performance under P-limited conditions. The QTL identified here have practical relevance because they were confirmed in the target genetic background of the local variety Nerica4 and can therefore be applied directly to improve its performance.

Comparative transcriptome analysis reveals a rapid response to phosphorus deficiency in a phosphorus-efficient rice genotype.

Phosphorus (P) is an essential plant nutrient. Most rice growing lands lack adequate P, requiring multiple P fertiliser applications to obtain expected yields. However, P fertiliser is environmentally damaging, and already unaffordable to the marginal farmers. This warrants developing P-efficient rice varieties that require less P to produce the expected yield. However, genetic factors underlying P-use efficiency (PUE) in rice remain elusive. Here, we conducted comparative transcriptome analysis using two rice varieties with contrasting PUE; a P-efficient landrace DJ123 and a P-inefficient modern cultivar IR64. We aimed to understand the transcriptomic responses in DJ123 that allow it to achieve a high PUE under low P conditions. Our results showed that both DJ123 and IR64 had replete tissue P concentrations after 48 h of P deprivation. Yet, DJ123 strongly responded to the external low P availability by inducing P starvation-inducible genes that included SPX2, PHO1, PAPs and SQDs, while these genes were not significantly induced in IR64. We envisage that the ability of DJ123 to rapidly respond to low P conditions might be the key to its high PUE. Our findings lay a valuable foundation in elucidating PUE mechanism in rice, thus will potentially contribute to developing P-efficient modern rice variety.

Genomic prediction of zinc-biofortification potential in rice gene bank accessions.

KEY MESSAGE: A genomic prediction model successfully predicted grain Zn concentrations in 3000 gene bank accessions and this was verified experimentally with selected potential donors having high on-farm grain-Zn in Madagascar. Increasing zinc (Zn) concentrations in edible parts of food crops, an approach termed Zn-biofortification, is a global breeding objective to alleviate micro-nutrient malnutrition. In particular, infants in countries like Madagascar are at risk of Zn deficiency because their dominant food source, rice, contains insufficient Zn. Biofortified rice varieties with increased grain Zn concentrations would offer a solution and our objective is to explore the genotypic variation present among rice gene bank accessions and to possibly identify underlying genetic factors through genomic prediction and genome-wide association studies (GWAS). A training set of 253 rice accessions was grown at two field sites in Madagascar to determine grain Zn concentrations and grain yield. A multi-locus GWAS analysis identified eight loci. Among these, QTN_11.3 had the largest effect and a rare allele increased grain Zn concentrations by 15%. A genomic prediction model was developed from the above training set to predict Zn concentrations of 3000 sequenced rice accessions. Predicted concentrations ranged from 17.1 to 40.2 ppm with a prediction accuracy of 0.51. An independent confirmation with 61 gene bank seed samples provided high correlations (r = 0.74) between measured and predicted values. Accessions from the aus sub-species had the highest predicted grain Zn concentrations and these were confirmed in additional field experiments, with one potential donor having more than twice the grain Zn compared to a local check variety. We conclude utilizing donors from the aus sub-species and employing genomic selection during the breeding process is the most promising approach to raise grain Zn concentrations in rice.

Phenotyping of a rice (Oryza sativa L.) association panel identifies loci associated with tolerance to low soil fertility on smallholder farm conditions in Madagascar.

Rice (Oryza sativa L.) is a staple food of Madagascar, where per capita rice consumption is among the highest worldwide. Rice in Madagascar is mainly grown on smallholder farms on soils with low fertility and in the absence of external inputs such as mineral fertilizers. Consequently, rice productivity remains low and the gap between rice production and consumption is widening at the national level. This study evaluates genetic resources imported from the IRRI rice gene bank to identify potential donors and loci associated with low soil fertility tolerance (LFT) that could be utilized in improving rice yield under local cultivation conditions. Accessions were grown on-farm without fertilizer inputs in the central highlands of Madagascar. A Genome-wide association study (GWAS) identified quantitative trait loci (QTL) for total panicle weight per plant, straw weight, total plant biomass, heading date and plant height. We detected loci at locations of known major genes for heading date (hd1) and plant height (sd1), confirming the validity of GWAS procedures. Two QTLs for total panicle weight were detected on chromosomes 5 (qLFT5) and 11 (qLFT11) and superior panicle weight was conferred by minor alleles. Further phenotyping under P and N deficiency suggested qLFT11 to be related to preferential resource allocation to root growth under nutrient deficiency. A donor (IRIS 313-11949) carrying both minor advantageous alleles was identified and crossed to a local variety (X265) lacking these alleles to initiate variety development through a combination of marker-assisted selection with selection on-farm in the target environment rather than on-station as typically practiced.

Rice increases phosphorus uptake in strongly sorbing soils by intra-root facilitation.

Upland rice (Oryza sativa) is adapted to strongly phosphorus (P) sorbing soils. The mechanisms underlying P acquisition, however, are not well understood, and models typically underestimate uptake. This complicates root ideotype development and trait-based selection for further improvement. We present a novel model, which correctly simulates the P uptake by a P-efficient rice genotype measured over 48 days of growth. The model represents root morphology at the local rhizosphere scale, including root hairs and fine S-type laterals. It simulates fast- and slowly reacting soil P and the P-solubilizing effect of root-induced pH changes in the soil. Simulations predict that the zone of pH changes and P solubilization around a root spreads further into the soil than the zone of P depletion. A root needs to place laterals outside its depletion- but inside its solubilization zone to maximize P uptake. S-type laterals, which are short but hairy, appear to be the key root structures to achieve that. Thus, thicker roots facilitate the P uptake by fine lateral roots. Uptake can be enhanced through longer root hairs and greater root length density but was less sensitive to total root length and root class proportions.

Below-ground plant-soil interactions affecting adaptations of rice to iron toxicity.

Iron toxicity is a major constraint to rice production, particularly in highly weathered soils of inland valleys in sub-Saharan Africa where the rice growing area is rapidly expanding. There is a wide variation in tolerance of iron toxicity in the rice germplasm. However, the introgression of tolerance traits into high-yielding germplasm has been slow owing to the complexity of the tolerance mechanisms and large genotype-by-environment effects. We review current understanding of tolerance mechanisms, particularly those involving below-ground plant-soil interactions. Until now these have been less studied than above-ground mechanisms. We cover processes in the rhizosphere linked to exclusion of toxic ferrous iron by oxidation, and resulting effects on the mobility of nutrient ions. We also cover the molecular physiology of below-ground processes controlling iron retention in roots and root-shoot transport, and also plant iron sensing. We conclude that future breeding programmes should be based on well-characterized molecular markers for iron toxicity tolerance traits. To successfully identify such markers, the complex tolerance response should be broken down into its components based on understanding of tolerance mechanisms, and tailored screening methods should be developed for individual mechanisms.

Breeding rice for a changing climate by improving adaptations to water saving technologies.

Climate change is expected to increasingly affect rice production through rising temperatures and decreasing water availability. Unlike other crops, rice is a main contributor to greenhouse gas emissions due to methane emissions from flooded paddy fields. Climate change can therefore be addressed in two ways in rice: through making the crop more climate resilient and through changes in management practices that reduce methane emissions and thereby slow global warming. In this review, we focus on two water saving technologies that reduce the periods lowland rice will be grown under fully flooded conditions, thereby improving water use efficiency and reducing methane emissions. Rice breeding over the past decades has mostly focused on developing high-yielding varieties adapted to continuously flooded conditions where seedlings were raised in a nursery and transplanted into a puddled flooded soil. Shifting cultivation to direct-seeded rice or to introducing non-flooded periods as in alternate wetting and drying gives rise to new challenges which need to be addressed in rice breeding. New adaptive traits such as rapid uniform germination even under anaerobic conditions, seedling vigor, weed competitiveness, root plasticity, and moderate drought tolerance need to be bred into the current elite germplasm and to what extent this is being addressed through trait discovery, marker-assisted selection and population improvement are reviewed.

Leaf phosphorus fractionation in rice to understand internal phosphorus-use efficiency.

BACKGROUND AND AIMS: Phosphorus (P) availability is often limiting for rice (Oryza sativa) production. Improving internal P-use efficiency (PUE) is crucial to sustainable food production, particularly in low-input systems. A critical aspect of PUE in plants, and one that remains poorly understood, is the investment of leaf P in different chemical P fractions (nucleic acid-P, lipid-P, inorganic-P, metabolite-P and residual-P). The overarching objective of this study was to understand how these key P fractions influence PUE. METHODS: Three high-PUE and two low-PUE rice genotypes were grown in hydroponics with contrasting P supplies. We measured PUE, total P, P fractions, photosynthesis and biomass. KEY RESULTS: Low investment in lipid-P was strongly associated with increased photosynthetic PUE (PPUE), achieved by reducing total leaf P concentration while maintaining rapid photosynthetic rates. All low-P plants exhibited a low investment in inorganic-P and lipid-P, but not nucleic acid-P. In addition, whole-plant PUE was strongly associated with reduced total P concentration, increased biomass and increased preferential allocation of resources to the youngest mature leaves. CONCLUSIONS: Lipid remodelling has been shown in rice before, but we show for the first time that reduced lipid-P investment improves PUE in rice without reducing photosynthesis. This presents a novel pathway for increasing PUE by targeting varieties with reduced lipid-P investment. This will benefit rice production in low-P soils and in areas where fertilizer use is limited, improving global food security by reducing P fertilizer demands and food production costs.

Corrigendum: QTL mapping for early root and shoot vigor of upland rice (Oryza sativa L.) under P deficient field conditions in Japan and Madagascar.

[This corrects the article DOI: 10.3389/fpls.2022.1017419.].

From gene banks to farmer's fields: using genomic selection to identify donors for a breeding program in rice to close the yield gap on smallholder farms.

KEY MESSAGE: Despite phenotyping the training set under unfavorable conditions on smallholder farms in Madagascar, we were able to successfully apply genomic prediction to select donors among gene bank accessions. Poor soil fertility and low fertilizer application rates are main reasons for the large yield gap observed for rice produced in sub-Saharan Africa. Traditional varieties that are preserved in gene banks were shown to possess traits and alleles that would improve the performance of modern variety under such low-input conditions. How to accelerate the utilization of gene bank resources in crop improvement is an unresolved question and here our objective was to test whether genomic prediction could aid in the selection of promising donors. A subset of the 3,024 sequenced accessions from the IRRI rice gene bank was phenotyped for yield and agronomic traits for two years in unfertilized farmers' fields in Madagascar, and based on these data, a genomic prediction model was developed. This model was applied to predict the performance of the entire set of 3024 accessions, and the top predicted performers were sent to Madagascar for confirmatory trials. The prediction accuracies ranged from 0.10 to 0.30 for grain yield, from 0.25 to 0.63 for straw biomass, to 0.71 for heading date. Two accessions have subsequently been utilized as donors in rice breeding programs in Madagascar. Despite having conducted phenotypic evaluations under challenging conditions on smallholder farms, our results are encouraging as the prediction accuracy realized in on-farm experiments was in the range of accuracies achieved in on-station studies. Thus, we could provide clear empirical evidence on the value of genomic selection in identifying suitable genetic resources for crop improvement, if genotypic data are available.

Cost-Benefit Analysis of the Upland-Rice Root Architecture in Relation to Phosphate: 3D Simulations Highlight the Importance of S-Type Lateral Roots for Reducing the Pay-Off Time.

The rice root system develops a large number of nodal roots from which two types of lateral roots branch out, large L-types and fine S-types, the latter being unique to the species. All roots including S-types are covered by root hairs. To what extent these fine structures contribute to phosphate (P) uptake under P deficiency was investigated using a novel 3-D root growth model that treats root hairs as individual structures with their own Michaelis-Menten uptake kinetics. Model simulations indicated that nodal roots contribute most to P uptake followed by L-type lateral roots and S-type laterals and root hairs. This is due to the much larger root surface area of thicker nodal roots. This thickness, however, also meant that the investment in terms of P needed for producing nodal roots was very large. Simulations relating P costs and time needed to recover that cost through P uptake suggest that producing nodal roots represents a considerable burden to a P-starved plant, with more than 20 times longer pay-off time compared to S-type laterals and root hairs. We estimated that the P cost of these fine root structures is low enough to be recovered within a day of their formation. These results expose a dilemma in terms of optimizing root system architecture to overcome P deficiency: P uptake could be maximized by developing more nodal root tissue, but when P is growth-limiting, adding more nodal root tissue represents an inefficient use of the limiting factor P. In order to improve adaption to P deficiency in rice breeding two complementary strategies seem to exist: (1) decreasing the cost or pay-off time of nodal roots and (2) increase the biomass allocation to S-type roots and root hairs. To what extent genotypic variation exists within the rice gene pool for either strategy should be investigated.

Genetic and physiological traits for internal phosphorus utilization efficiency in rice.

Phosphorus (P) is an essential macronutrient for plant growth and development. Phosphorus is usually applied as fertilizer obtained from rock phosphate which is a non-renewable resource. Therefore, developing rice varieties that can use P more efficiently is crucial. Here, we investigated genotypic differences in traits related to internal Phosphorus Utilization Efficiency (PUE) in five rice genotypes grown under P-deficient conditions. P-efficient rice genotypes showed higher total biomass. This was partly due to higher root biomass, which in turn relied on preferential allocation of P to roots in these genotypes. Changes in P content and tissue P concentrations were analyzed in individual leaves at different time points. Genotypes belonging to the high-PUE group responded more quickly to P starvation in terms of reducing leaf P concentrations and they were able to reduce these concentrations to a lower level compared to the low-PUE group. Changes in P concentrations were reflected in gene expression levels for genes involved in lipid remodeling. Sulfolipid (OsSQD2) and galactolipid (OsMGD and OsDGD) synthesis-related genes were generally induced due to P starvation with most pronounced up-regulation in OsDGD1 and OsMGD3, but patterns differed between genotypes. A significantly higher expression of OsDGD5 and OsMGD1 & 2 was detected in the youngest fully expanded leaf of the high-PUE genotype group, whereas expression levels were reversed in older leaves. This pattern would confirm that P efficient genotypes react faster to P starvation in terms of freeing P for redistribution to growing tissues and replacing phospholipids with galactolipids in younger leaves may contribute to this aspect.

Metabolomic markers and physiological adaptations for high phosphate utilization efficiency in rice.

Utilizing phosphate more efficiently is crucial for sustainable crop production. Highly efficient rice (Oryza sativa) cultivars have been identified and this study aims to identify metabolic markers associated with P utilization efficiency (PUE). P deficiency generally reduced leaf P concentrations and CO2 assimilation rates but efficient cultivars were reducing leaf P concentrations further than inefficient ones while maintaining similar CO2 assimilation rates. Adaptive changes in carbon metabolism were detected but equally in efficient and inefficient cultivar groups. Groups furthermore did not differ with respect to partial substitutions of phospholipids by sulfo- and galactolipids. Metabolites significantly more abundant in the efficient group, such as sinapate, benzoate and glucoronate, were related to antioxidant defence and may help alleviating oxidative stress caused by P deficiency. Sugar alcohols ribitol and threitol were another marker metabolite for higher phosphate efficiency as were several amino acids, especially threonine. Since these metabolites are not known to be associated with P deficiency, they may provide novel clues for the selection of more P efficient genotypes. In conclusion, metabolite signatures detected here were not related to phosphate metabolism but rather helped P efficient lines to keep vital processes functional under the adverse conditions of P starvation.

Lateral Roots: Random Diversity in Adversity.

Lateral roots are essential for soil foraging and uptake of minerals and water. They feature a large morphological diversity that results from divergent primordia or root growth and development patterns. Besides a structured diversity, resulting from the hierarchical and developmental organization of root systems, there exists a random diversity, occurring between roots of similar age, of the same hierarchical order, and exposed to uniform conditions. The physiological bases and functional consequences of this random diversity are largely ignored. Here we review the evidence for such random diversity throughout the plant kingdom, present innovative approaches based on statistical modeling to account for such diversity, and set the list of its potential benefits in front of a variable and unpredictable soil environment.

Enhanced ascorbate level improves multi-stress tolerance in a widely grown indica rice variety without compromising its agronomic characteristics.

A biotechnological approach was adopted for increasing foliar ascorbate levels as a strategy to adapt a widely grown high yielding rice variety to multiple abiotic stresses. The variety IR64 (Oryza sativa L. ssp. indica) was engineered to express the ascorbate biosynthesis gene GDP-L-galactose phosphorylase (AcGGP) from kiwifruit (Actinidia chinensis Planch.) under the control of a leaf-specific promoter of the Leaf Panicle 2 (LP2) gene. Transgene expression increased foliar ascorbate levels up to >2.5 fold but did not lead to any changes in morphological traits (seed yield, sterility rate, grain weight, and biomass) in non-stress conditions. We then hypothesized that enhanced foliar ascorbate would confer multi-stress tolerance. Indeed transgenic lines were more tolerant to salinity in terms of lipid peroxidation and foliar symptoms, and to drought in terms of lipid peroxidation and post-drought recovery (number of dead leaves). A significantly better performance in ozone stress was seen only when ozone coincided with salinity. However, no differences between transgenic lines and wild types occurred when plants were subjected to toxicities in redox-active transition metals, i.e. iron and manganese, although plants showed clear symptoms of oxidative stress. Moreover, no differential response to zinc deficiency was observed, because the background genotype IR64 was not sensitive to this stress. Taken together, our study helps to identify stress conditions that can be mitigated by enhancing foliar ascorbate levels, and therefore facilitates an adaptive breeding approach for multiple stresses that would not imply any yield penalty.