Abiotic stress alleles harbored by Colombian cultivars shed light on adaptation mechanisms and needs for further genetic improvement
The rice cultivars Llanura11 and Porvenir12 are cultivated as tropical japonica upland varieties in the savannas of Colombia which form part of the great Savanna biome (Amezquita et al. 2013; Rincón et al. 2014; Saito et al. 2018). These ecosystems are characterized by highly acidic, low fertility soils associated with Al toxicity, P deficiency, and drought alternating with high precipitation. To investigate the potential for targeted genetic improvement, we characterized alleles found in these varieties at five major-effect genes known to confer Al toxicity tolerance (ART1), drought avoidance (DRO1), submergence tolerance (SUB1A), P-acquisition (PSTOL1), and P-use efficiency (SPDT) via PCR and sequencing. Desirable alleles were found at ART1, DRO1, and PSTOL1, suggesting that adaptative mechanisms associated with physiological response to soil pH and ionic composition, including root growth angle and overall root system architecture are conferred by these loci in response to drought-prone acidic soil environments. In fact, Llanura11, also known as Cirad 409 (Grenier C., personal communication), is characterized by a strong root system under irrigated conditions and an increased total root volume under drought conditions (Guimarães et al. 2020).
The fact that the Colombian cultivars carry Al tolerant alleles at ART1, a deep rooting allele at DRO1, and a likely P-deficiency tolerant allele at PSTOL1 is consistent with their adaptation to acid soils. In rice varieties that are susceptible to Al toxicity (a ubiquitous feature of low pH soils), root growth is severely limited (Famoso et al. 2011; Kochian et al. 2015), making it difficult for roots to explore the soil and reach deeper horizons to find water (Uga et al. 2013) and/or to find an immobile nutrient such as Pi (Heuer et al. 2017). Thus, favorable alleles at these three loci would be mutually complementary in promoting root growth in acid soils. The present study suggests that the desirable PSTOL1 allele found in the Colombian cultivars shares ancestry with the PSTOL1 allele found in CG14, a cultivar of African rice, O. glaberrima. This allele is different from the allele originally cloned from the aus variety Kalalath (Gamuyao et al. 2012; Pariasca-Tanaka et al. 2014), and while it appears to be favorable, it remains to be determined whether the CG14 allele confers a similar P-uptake advantage as the Kasalath allele in the cultivars under investigation.
In contrast to ART1, DRO1 and PSTOL1, adaptive alleles at SUB1A and SPDT were lacking, suggesting that targeted introgression and/or gene editing of favorable alleles at these loci could enhance the performance of Llanura11 and Porvenir12. The SUB1 locus is known to harbor a gene family consisting of 2–3 tandemly arrayed members on chromosome 9 (Fukao et al. 2006), with the SUB1A family member conferring tolerance to complete submergence in the aus variety FR13A for up to 14 days (Xu et al. 2006; Singh et al. 2020). It is also associated with enhanced tolerance to other abiotic stresses, including drought (Fukao et al. 2011; Bin Rahman and Zhang 2016). The SUB1A gene is absent in the Nipponbare reference genome and other japonica varieties due to an inversion and deletion (Singh et al. 2010). Consistent with this, we found that the gene was absent from the tropical japonica Llanura11 and Porvenir12 cultivars (Fig. 2). Although the Colombian savannas are characterized by a strong dry season, there is also an important water surplus due to precipitation and even floods (Rincón et al. 2014), suggesting that marker-assisted introgression of SUB1A may be the most efficient way to generate climate-resilient varieties for these environments.
SPDT, a gene involved in internal P use efficiency (PUE), was cloned in cv. Nipponbare by transposon Tos17 tagging (Yamaji et al. 2017). The loss-of-function desirable allele causes less P to be allocated to the grain (by about 20%) and consequently a potential increase of bioavailability of essential nutrients (Fe, Zn) without affecting seed germination and grain yield (Yamaji et al. 2017). At the same time, more P is assigned to the straw, which remains in the field after harvest and could be used as a fertilizer (Rose et al. 2013; Yamaji et al. 2017). The sequence of this gene locus in the upland cultivars under investigation showed that the wild-type allele was present. Thus, further cultivar improvement by knocking out SPDT offers an interesting possibility to generate high PUE and grain quality cultivars.
Given that directed DNA insertion is both technically challenging and presents regulatory hurdles (because it would be subjected to the restrictions imposed on genetically modified organisms (GMOs) (Georges and Ray 2017; Wang et al. 2017), it would be advisable to introgress the SUB1A gene into the two Colombian varieties using marker-assisted selection (MAS). This approach has successfully introduced submergence tolerance into varieties grown on thousands of hectares in flood-prone regions of Asia and Africa (Mickelbart et al. 2015). On the other hand, editing the SPDT gene through a targeted gene deletion offers a practical solution, given that a DNA-free editing strategy could be used, posing fewer technical and regulatory constraints (Chilcoat et al. 2017; Georges and Ray 2017).
The SPDT phenotype was next examined in the Colombian cultivars and compared with Nipponbare (SPDT wt) and the two Tos17 mutant lines (spdt). This was done by evaluating gene expression (using qPCR) and organ P concentration at the early vegetative and seed maturity stages. For qPCR, the SBR was targeted for analysis based on a report showing that this tissue produced the most significant SPDT differential expression using 0 and 90 µM P treatments under hydroponic conditions (Yamaji et al. 2017). The induction of SPDT expression under 0 µM P in the reference Nipponbare was 2.3-fold higher compared to the control condition of 90 µM P (Fig. 4c). This represents a smaller induction of gene expression as compared to that observed in the previous study for Nipponbare (6.5-fold increase) (Yamaji et al. 2017). There was also no difference in P concentration in the youngest leaf 8 at the 8-leaf stage (Fig. 4b) as opposed to the reported in theprevious study (Yamaji et al. 2017), where lower P concentrations were detected in the sdpt mutants as compared to wild type Nipponbare. The differences in gene expression and P concentrations in the 8th leaf in the two studies are likely due to differences in the nutrient solutions used (Magnavaca in the present study vs. Kimura in Yamaji et al. (2017) (Table S3)) and/or small differences in experimental conditions.
Llanura11 and Porvenir12 were most similar to Nipponbare in terms of induced RNA expression under P-deficiency conditions, increased P concentration in older leaves during early vegetative development (5 and 8- leaf stages) and in flag and green leaves at seed maturity, and reduced P concentrations in BS (Figs. 4–5). Based on these results, we concluded that the two Colombian cultivars carry the wild-type SPDT allele and are therefore likely to benefit from deletion of the SULTR3;4 gene, mimicking the results observed in the ND0047 and NE3502 mutant lines.
Early phenotyping for fast screening of spdt genotypes
The SPDT phenotype was previously evaluated at the 8-leaf stage using Kimura B solution at half-strength (Yamaji et al. 2017). One objective of this study was to determine whether we could phenotypically differentiate the SPDT wild-type from the spdt knock-out lines at earlier stages of development, i.e., at the 5-leaf stage in plants growin in Magnavaca solution (Famoso et al. 2011), which has 7.5 times greater ionic strength than the Kimura B½ solution (Table S3). We also grew plants in a growth chamber rather than in a greenhouse which helped to minimize environmental variation, providing greater consistency of temperature and luminosity, regardless of the time of year.
Our results indicate that the earlier 5- leaf stage of development and higher ionic strength solution under growth chamber conditions can be used to reliably evaluate the phenotype under hydroponic conditions, saving time and resources (Fig. 4). The original method required 32–42 days to evaluate the Nipponbare phenotype at the 8- or 9-leaf stage (Yamaji et al. 2017), while the modified method required 22 days for Nipponbare, and 25 days for the Colombian varieties to reach the 5-leaf stage. Nipponbare takes 33 days and the Colombian varieties 42 days to develop to the 8-leaf stage in the Magnavaca solution under growth chamber conditions (Figure S3). The higher ionic strength of the growth solution, a high volume ratio (about 10 liters of solution/plant), and shorter growth periods resulted in minimal nutrient depletion from the solutions, as indicated by routinary ICP analysis of the Magnavaca solution throughout the experiment. These modifications reduce water usage, minimal replacement of microelements, and overall less labor. Therefore, this modified protocol represents an improved technique to quickly screen for desirable spdt mutants at the seedling stage.
Low P brown seed as a potential source of bioavailable nutrients
Varieties that transfer less P to developing grains offer environmental and nutritional benefits (Rose et al. 2013). Phytic acid salt (phytate) is the main form of P in cereal grains where it acts as an antinutrient, decreasing the bioavailability of other essential nutrients, such as Fe and Zn (Perera et al., 2018). Phytate accumulates in the bran or aleurone layer of brown rice seed where it can chelate Fe, K, and Ca, while Zn is found broadly distributed from the aleurone layer to the inner endosperm, often bound loosely to phytic acid but also found in another storage form (Iwai et al. 2012). In this study, we investigated the hypothesis that reducing this chelant might increase nutrient bioavailability and nutritional benefits by comparing P, Fe, and Zn concentrations of BS and PS in spdt mutants and wild-type rice.
We confirmed that higher P concentrations were detected in BS in SPDT wild-type genotypes (Nipponbare and Colombian cultivars) compared with spdt mutants, while no differences were found in PS for either P or phytate concentrations, nor for Fe bioavailability (Fig. 5).
This lack of correspondence between Fe concentration and Fe uptake determined by the caco-2 cell bioavailability assay has been previously reported for brown rice (Glahn et al. 2002).
The molar ratios of Phytic acid:Fe were very high (Table S5), indicating that not much Fe in the PS is contributing from a nutritional perspective. This is in agreement with the fact that only 4.335 ng of ferritin/mg of protein was formed in the control Nipponbare with no significant differences among genotypes (Fig. 5e), therefore, not much Fe is likely to be delivered from the PS. On the other hand, the molar ratios of phytate:Zn were much lower (Figure S5). Typical Zn levels in polished rice are low (8–12 ppm) but there is a wide genetic variability in brown (7.3 to 52.7 ppm) and polished (8 to 38 ppm) rice (Babu et al. 2020). The primary inhibitor of Zn is phytate (Lönnerdal 2000) and more Zn, as observed in the mutans and Colombian genotypes of this study (Fig. 5f), usually means more absorbed Zn. However, to our knowledge there is not a good method for assessing Zn bioavailability.
Brown or unmilled rice is known to have higher vitamin and mineral content compared to milled rice (Muthayya et al. 2014). The present study confirms higher Fe and Zn concentrations in BS compared to PS. However, many of the nutritional benefits of brown rice cannot be not realized if nutrient bioavailability is compromised by high levels of phytate in BS. Thus, low P spdt mutants that accumulate less phytate in the bran offer a possible solution for breeders interested in developing cereal varieties with high concentrations of bioavailable Fe and Zn as part of the human diet and also as a component of animal feed. Low phytate grain would be particularly useful in non-ruminant livestock feeds, including poultry, swine, and fish feeds, where P and inositol in phytic acid are generally not bioavailable, and P-deficiency is a problem, while at the same time it may help reduce P excretion that contributes to environmental problems, such as eutrophication of waterways (Perera et al. 2018).
Technical, regulatory, and societal implications of editing the SPDT gene
P is an essential micronutrient, and P-deficiency is a major constraint for crop yield; thus, to obtain high yields, regular applications of P- fertilizer are needed, but the supply of phosphate rock is limited (Rose et al. 2013). P efficiency (PE), the capacity of plants to tolerate stress caused by P-deficiency, can be achieved by increasing P acquisition efficiency (PAE) or PUE traits (Rose et al. 2013; Heuer et al. 2017). The latter has prompted more recent attention and has been envisaged that breeding crops by lowering grain P concentration is one approach to increase PUE in cereal systems since less P removed from the field could lower fertilizer requirements, saving production costs to farmers (Rose et al. 2013). For example, the low phytic acid (lpa) recessive mutation lpa1-1 in barley, harbored by the US commercial cultivar “Herald”, reduces total grain P by 10–20% and lowers phytate levels without a penalty on subsequent crop yields (Bregitzer et al. 2007; Ye et al. 2011).
In rice, the genetic architecture of PUE was investigated using GWAS with a rice diversity panel grown in a hydroponic system to ensure uniform access to P (Wissuwa et al. 2015). The study identified loci associated with PUE on four chromosomes, with the chromosome 1 haplotype showing high priority based on association with candidate genes of potential utility in plant breeding. Alternatively, novel variation can be generated by mutation breeding (Rose et al. 2013; Heuer et al. 2017). In fact, transporters involved in delivering phosphate to developing seeds and synthesis of phytic acid have been discovered through mutation screens, identifying key genes belonging to the Sulfate Transporter (SULTR) family. One of them, OsSULTR3;3, was discovered in two lpa mutants developed to improve the nutritional value of rice grains. Disruption of OsSULTR3;3 in these mutants leads to reduced concentrations of total grain P (19–28%) and grain phytate (35–45%) (Zhao et al. 2016), with the rice SULTR3;3 gene is closely related to the barley sulfate transporter LPA1 (Ye et al. 2011; Zhao et al. 2016). More recently, OsSULTR3;4 (referred to as SPDT in this study), the first characterized transporter for inorganic P in this family, was shown to be involved in P allocation to rice grain, with the desired mutation reducing P (by 20%) as well as reducing phytate concentration. These discoveries point to a potential role in improving PUE in rice cropping systems such that disruption of the Pi transporter leads to retention of P in the straw, that it can be easily returned to the field after harvest (as a form of mulch) to help fertilize the next season’s crop (Yamaji et al. 2017).
Here we aim to leverage knowledge about SPDT to enhance PUE in two upland Colombian cultivars using CRISPR-associated endonuclease as a targeted form of mutagenesis. This system has great precision and minimal risk of introducing off-target variation in the genome compared to historical mutagenesis techniques (Graham et al. 2020). We generated specialized reagents and protocols to generate an SPDT deletion using CRISPR/Cas9 in the temperate japonica variety, Nipponbare, and tropical japonica upland cultivars, Llanura11 and Porvenir12, as a foundation for the strategy. The reagents included plant transformation vectors containing the Cas9 and gRNAs to target identical SPDT regions in the three cultivars. We confirmed the intended deletion of 7.3 Kb at the SPDT locus by transfecting Nipponbare protoplasts with a vector targeting the 5’ and 3’ up- and downstream gene ends (Fig. 6). The protocols included regeneration and transformation methods mediated by Ribonucleoprotein complexes (RNP) and Agrobacterium tumefaciens in the three cultivars. Reagents and protocols are essential tools for proof-of-concept to determine whether SPDT can be subjected to a targeted deletion in cultivars of interest; whether desired edits, if obtained in plants, produce the improved phenotype to enhance PUE and grain quality under acid soils; and whether individual effects of P reduction in the grain-based on deletions of SULTR3;3 and SULTR3;4 can be leveraged by pyramiding mutations in both genes in a single cultivar.
Our work establishes the basis for targeted deletion of SPDT in upland rice cultivars of interest. Gene deletions are preferred over gene insertions because they tend to present fewer societal and regulatory concerns. Currently, genome edits involving deletions are usually classified into the Site Directed Nuclease − 1 (SDN-1) category, as long as there is no addition of foreign DNA (Schmidt et al. 2020). SDN-1 edits follow the standards of conventional mutagenesis with categorization based on the product and are considered non-regulated as GMOs in most countries except the European Union and New Zealand (Schmidt et al. 2020). The first Xanthomonas oryzae pv. oryzae (Xoo) genome-edited resistant rice, where promoter elements of the sucrose transporter genes SWEET were targeted through CRISPR/Cas9 (Oliva et al. 2019) was declared transgene-free, non-regulated, and equivalent to what could be accomplished with conventional breeding in Colombia and the USA (Agdaily 2020). This opens the path for approval of other DNA-free editing products such as those proposed in this study for the Colombian savannas.
In addition, the fact that we developed protocols for RNP mediated transformation as a backup to Agrobacterium transformation aims to further ease regulatory and societal concerns. RNP consists of in vitro transcribed gRNAs in complex with the Cas9 protein and is a DNA-free editing method where no foreign DNA is used, and therefore the edits obtained are completely transgene-free (Zhang et al. 2016; Liang et al. 2017). Moreover, the CRISPR/Cas9 RNP complex can be quickly degraded in vivo. Thus, the genome is less prone to off-target mutations as compared to editing through Agrobacterium transformation where the CRISPR/Cas9 DNA construct is incorporated in the genome (Liang et al. 2017). This work lays the foundation for generating a DNA-free edited cultivar, either by using the RNP approach directly or by eliminating the transgene via segregation in edited plants transformed using Agrobacterium. Further investigation is needed to determine which approach will be the most appropriate in terms of technical feasibility, efficiency, and regulatory and societal concerns as we continue our efforts to generate SPDT-edited lines in different genetic backgrounds to improve P-acquisition and P-use efficiency in the upland rice ecosystem in the Colombian savannas.