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Impact of rice GENERAL REGULATORY FACTOR14h (GF14h) on low-temperature seed germination and its application to breeding [1]

['Yusaku Sugimura', 'Iwate Biotechnology Research Center', 'Kitakami', 'Iwate', 'Kaori Oikawa', 'Yu Sugihara', 'Crop Evolution Laboratory', 'Kyoto University', 'Muko', 'Kyoto']

Date: 2024-08

Direct seeding is employed to circumvent the labor-intensive process of rice (Oryza sativa) transplantation, but this approach requires varieties with vigorous low-temperature germination (LTG) when sown in cold climates. To investigate the genetic basis of LTG, we identified the quantitative trait locus (QTL) qLTG11 from rice variety Arroz da Terra, which shows rapid seed germination at lower temperatures, using QTL-seq. We delineated the candidate region to a 52-kb interval containing GENERAL REGULATORY FACTOR14h (GF14h) gene, which is expressed during seed germination. The Arroz da Terra GF14h allele encodes functional GF14h, whereas Japanese rice variety Hitomebore harbors a 4-bp deletion in the coding region. Knocking out functional GF14h in a near-isogenic line (NIL) carrying the Arroz da Terra allele decreased LTG, whereas overexpressing functional GF14h in Hitomebore increased LTG, indicating that GF14h is the causal gene behind qLTG11. Analysis of numerous Japanese rice accessions revealed that the functional GF14h allele was lost from popular varieties during modern breeding. We generated a NIL in the Hitomebore background carrying a 172-kb genomic fragment from Arroz da Terra including GF14h. The NIL showed superior LTG compared to Hitomebore, with otherwise comparable agronomic traits. The functional GF14h allele from Arroz da Terra represents a valuable resource for direct seeding in cold regions.

Rice serves as a fundamental crop sustaining over half of the global population. With the rapid growth of the world’s population, it will become increasingly important to improve rice productivity. On the other hand, the aging of rice farmers in Japan has resulted in a constant labor shortage. To address this, direct seeding, in which seeds are sown directly in rice fields without going through the most labor-intensive part of the rice cultivation process, i.e., seedling production and transplanting, has been recommended. However, prevalent elite rice varieties are known to be unsuitable for direct seeding due to their poor seed germination ability under low-temperature conditions. In this study, we show for the first time that GF14h gene from the Portuguese variety Arroz da Terra improves seed germination at low temperatures (LTG). In addition, a novel cross-bred line was generated by introducing the GF14h-containing genomic segment from Arroz da Terra into Hitomebore, a widely cultivated variety in northern Japan. This line is expected to be used as a pre-breeding material to enhance LTG. This study will provide a genetic basis for LTG and contribute to basic and applied research progress.

Funding: This work was partly supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement, IVG3007), received by HT, RT and AA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The NGS raw reads were deposited in the DNA Data Bank of Japan (DDBJ) under the BioProject accession numbers PRJDB13449, PRJDB13450, PRJDB13864, and PRJDB17450. Supplementary Tables S2 and S5 include the SRA accession numbers. The genome sequences assembled in this study were deposited at Zenodo ( https://doi.org/10.5281/zenodo.10460309 ). All relevant data are within the paper and its Supporting Information files.

Copyright: © 2024 Sugimura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

QTL pyramiding has been proposed as a breeding concept [ 29 ] for bringing together several QTLs (or genes) related to agronomically important traits in the genetic background of locally adapted elite cultivars. In practice, it is essential to generate pre-breeding materials for QTL pyramiding, i.e., near-isogenic lines (NILs) that harbor one or a few genomic segments introgressed from the donor parent into the genome of the recipient parent through a combination of continuous backcrossing and selfing via marker-assisted selection [ 30 ]. In this study, we determined that GF14h is responsible for an LTG-related QTL in Portuguese rice variety Arroz da Terra. We generated a NIL in the background of rice cultivar Hitomebore, which is adapted for growth in northern Japan, by replacing its GF14h genomic fragment with that from Arroz da Terra and tested its LTG performance.

14-3-3 proteins are regulatory proteins that are widely conserved in eukaryotes. These proteins bind to phosphorylated serine and tyrosine residues in their target proteins that participate in signal transduction and the regulation of gene expression [ 21 , 22 ], thus altering their enzymatic activity, subcellular localization, stability, or protein–protein interactions [ 23 – 25 ]. The rice genome encodes eight 14-3-3 proteins, named GF14a–h for GENERAL REGULATORY FACTOR14 [ 26 ]. GF14h is involved in rice seed germination under optimal temperature conditions [ 27 , 28 ]. In addition, GF14h contributes to phytohormone signaling, including abscisic acid and gibberellin signaling [ 27 , 28 ]. However, it remains unclear whether GF14h promotes seed germination under low-temperature conditions [ 28 ].

LTG is a quantitative trait regulated by complex molecular mechanisms. Linkage mapping and genome-wide association studies (GWAS) have identified over 30 LTG-related quantitative trait loci (QTLs) or genomic regions associated with this trait, located on all 12 rice chromosomes [ 3 – 20 ]. However, only a few genes involved in LTG have been described, such as qLTG3-1 [ 3 ] and STRESS-ASSOCIATED PROTEIN16 (OsSAP16) [ 15 ]. The qLTG3-1 gene, encoding a protein of unknown function, has a substantial influence on LTG [ 3 ]. During seed germination, qLTG3-1 expression is strongly induced in embryos, which leads to the loosening of the tissues covering the embryo by promoting vacuolation [ 3 ]. OsSAP16 encodes a stress-associated protein with two AN1-C2H2 zinc finger domains [ 15 ]. OsSAP16 presumably acts as a regulator of LTG.

Low-temperature seed germination (LTG) is a pivotal agronomic trait in rice (Oryza sativa). As rice originated from tropical and subtropical regions, it is highly susceptible to low-temperature conditions compared to other cereal crops such as wheat (Triticum aestivum) and barley (Hordeum vulgare) [ 1 ]. Nevertheless, rice is produced in temperate and high-altitude regions, where it frequently experiences temperatures below 20°C. In Japan, rice is abundantly cultivated in relatively cold areas such as Tohoku and Hokkaido. In recent years, there has been an increasing demand to shift from conventional transplantation-based rice cultivation to direct seeding to reduce labor and costs. However, direct seeding raises the risk of exposure to low temperatures during seed germination [ 2 ]. Therefore, to expand the use of direct seeding, it is crucial to breed rice cultivars with enhanced LTG.

Results

Identification of GF14h as the candidate gene for qLTG11 To delineate the qLTG11 region, we carried out map-based cloning using a segregating population derived from a cross between BC 2 F 3 line qLTG11-NIL and Japanese elite cultivar Hitomebore (S3A Fig). For mapping, we conducted germination tests at 15°C. We narrowed down the genomic region containing the QTL to a 52-kb segment (from 23.512 bp to 23.564 Mb) on chromosome 11 based on the Nipponbare reference genome (IRGSP-1.0) (Fig 2A). This interval contains two annotated genes based on the Nipponbare genome sequence (Fig 2B). We compared the genomic sequence of Hitomebore and Arroz da Terra across the candidate region using de novo genome assembly obtained from Nanopore long reads. The cultivars Hitomebore and Nipponbare had an identical genomic sequence over the entire candidate region (S6A Fig). By contrast, the genome sequence from Arroz da Terra was substantially different from that of Nipponbare, with the equivalent candidate region spanning approximately 94 kb (Figs 2B and S6B). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Positional cloning of qLTG11. (A) Fine mapping of qLTG11 to a 52-kb region between markers E and J. The chromosomal positions are based on the Nipponbare reference genome (Os-Nipponbare-Reference-IRGSP-1.0). Germination percentage was determined at 9 days of incubation at 15°C. Red and blue rectangles indicate chromosomal segments homozygous for Arroz da Terra or Hitomebore, respectively. Different lowercase letters indicate significant differences (n = 3 biologically independent samples, P < 0.001, Tukey’s HSD test). (B) Genomic structure of the candidate genomic region in Arroz da Terra and Hitomebore. Os11g0609600 (shown in red), encoding GF14h, is expressed in germinating seeds. (C) Diagram of the GF14h gene structure and sequence polymorphisms between Arroz da Terra and Hitomebore. The chromosomal positions are based on the Nipponbare reference genome. The coding region of GF14h in Hitomebore is identical to that in Nipponbare. The 4-bp deletion in Hitomebore causes a frameshift and the introduction of a premature stop codon. (D) Relative GF14h expression levels in germinating seeds of qLTG11-NIL. This expression analysis was conducted by RT-qPCR. In the boxplots, the box edges represent the upper and lower quantiles, the horizontal line in the middle of the box represents the median value, whiskers represent the lowest quantile to the top quantile, and the black squares show the mean. Five biological replicates were measured independently. Different lowercase letters indicate significant differences based on Tukey’s HSD test (P < 0.05). OsActin1 (Os03g0718100) was used for normalization. https://doi.org/10.1371/journal.pgen.1011369.g002 As the causal gene behind the variation in LTG is likely expressed in seeds, we performed transcriptome deep sequencing (RNA-seq) during seed germination in Hitomebore and qLTG11-NIL (S1 Table). Within the candidate region, the gene Os11g0609600, corresponding to the 14-3-3 gene GF14h, was expressed in both Hitomebore and qLTG11-NIL, whereas Os11g0609500 (Jacalin-like lectin domain containing protein) was not expressed in seeds (S7 Fig), thus suggesting that GF14h is a strong candidate gene for LTG. The GF14h gene structure and haplotypes in Arroz da Terra and Hitomebore are shown in Fig 2C. We detected a 4-bp deletion in the GF14h coding region in Hitomebore, causing a frameshift mutation predicted to introduce a premature stop codon (Figs 2C and S8). These results suggest that Hitomebore carries a loss-of-function allele of GF14h. To assess the role of GF14h in LTG, we examined the expression pattern of the putative functional GF14h (GF14hArroz) allele during seed germination at low temperature (15°C) using qLTG11-NIL. RT-qPCR analysis of GF14h expression levels showed that they were comparable in the embryo and endosperm at 1 and 3 days after the onset of seed imbibition (Fig 2D). At the beginning of germination, when a white coleoptile was visible (5 and 7 days after seed imbibition), GF14h expression levels rose in the endosperm, but not in the embryo (Fig 2D). By nine days of imbibition, when most seeds had germinated, GF14h expression in the endosperm returned to basal levels (Fig 2D). These results support the notion that GF14h plays a role in seed germination at low temperature.

GF14h plays a vital role in LTG To investigate the contribution of GF14h to LTG, we knocked out the functional GF14h copy present in qLTG11-NIL by clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing and evaluated LTG. Specifically, we introduced two single guide RNA (sgRNA) constructs targeting the exons of GF14h individually into qLTG11-NIL by Agrobacterium-mediated transformation. We chose to knock out GF14h in the qLTG11-NIL background rather than Arroz da Terra to evaluate the specific contribution of GF14h to LTG without the influence of qLTG3-1, which would be present in the Arroz da Terra background. To accurately evaluate the phenotypes of the edited plants, we selected heterozygous plants in the T 0 generation and isolated homozygous mutant lines and their unedited homozygous siblings in the T 1 generation. We obtained four knockout lines (gf14h-1, gf14h-2, gf14h-3, and gf14h-4) and their wild-type sibling (WTArroz) (S9 Fig). We detected significant drops in the germination percentage in all four knockout lines compared to WTArroz (Fig 3A and 3B). We also found that the knockout lines tended to have lower germination rates than WTArroz under normal temperature conditions (25°C) (S10 Fig), which is consistent with the previous report [27]. Furthermore, we generated transgenic lines in the Hitomebore background overexpressing the functional GF14h allele from Arroz da Terra under the control of the CaMV 35S promoter. In these overexpression lines, GF14h expression increased approximately 1,000-fold compared to the wild-type sibling (WTHitomebore) (S11 Fig). Importantly, the overexpression lines showed higher LTG than WTHitomebore when tested at 15°C (Fig 3C and 3D). Taken together, these data indicate that GF14h is a key gene involved in LTG. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Effect of GF14h mutation and overexpression on low-temperature germination. (A) Representative photographs showing seed germination in wild-type harboring Arroz-type GF14h (WTArroz) and CRISPR/Cas9 knockout lines (gf14-1) at 8 days after the onset of seed imbibition. Scale bar, 1 cm. (B) Seed germination rate of WTArroz and its CRISPR/Cas9 knockout lines at 7 days of seed imbibition at 15°C. The two target constructs (S9 Fig) were introduced into the qLTG11-NIL line. Data are means ± standard error (SE, n = 3). Different lowercase letters indicate significant differences based on Tukey’s HSD test (P < 0.01). (C) Representative photographs showing seed germination of wild-type (WTHitomebore) and OsGF14hArroz overexpression lines (OsGF14hArroz-Ox #2) at 7 days of seed imbibition at 15°C. Scale bar, 1 cm. (D) Seed germination rate of WTHitomebore and GF14hArroz overexpression lines in the Hitomebore background at 7 days of seed imbibition at 15°C. Data are means ± SE (n = 3). Different lowercase letters indicate significant differences based on Tukey’s HSD test (P < 0.05). https://doi.org/10.1371/journal.pgen.1011369.g003

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011369

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