Abstract

   The abounding variations in wild rice provided potential reservoirs of
   beneficial genes for rice breeding. Maintaining stable and high yields
   under environmental stresses is a long-standing goal of rice breeding
   but is challenging due to internal trade-off mechanisms. Here, we
   report wild rice GL12^W improves grain length and salt tolerance in
   both indica and japonica genetic backgrounds. GL12^W alters cell length
   by regulating grain size related genes including GS2, and positively
   regulates the salt tolerance related genes, such as NAC5, NCED3, under
   salt stresses. We find that a G/T variation in GL12 promoter determined
   its binding to coactivator GIF1 and transcription factor WRKY53. GIF1
   promotes GL12^W expression in young panicle and WRKY53 represses GL12^W
   expression under salt stresses. The G/T variation also contributes to
   the divergence of indica and japonica subspecies. Our results provide
   useful resources for modern rice breeding and shed insights for
   understanding yield and salt tolerance trade-off mechanism.

   Subject terms: Gene regulation, Plant breeding, Agricultural genetics,
   Abiotic
     __________________________________________________________________

   Maintaining yield under environmental stresses is a long-standing but
   challenging goal of rice breeding. Here, the authors report a MYB
   transcription factor encoding gene GL12 from wild rice can improve
   grain length and salt tolerance in cultivated rice and the
   identification of its upstream regulators GIF1 and WRKY53.

Introduction

   The domestication of cultivated rice is considered as one of the most
   important events in human agricultural history^[70]1. Currently
   cultivated rice (Oryza sativa) is a staple food that feeds more than
   half of the world’s population^[71]2.To meet the growing demand for
   rice production, it is essential to cultivate high-yielding and
   environmentally resilient rice varieties. However, simultaneous
   improvement of yield and biotic/abiotic resistance remains a huge
   challenge for rice breeders because yield and stress tolerance are
   typically negatively correlated^[72]2,[73]3.

   Grain size and shape are major determinants for grain yield and
   quality, and have been selected during early rice domestication^[74]4.
   Grain size and shape are quantitative traits regulated by a series of
   quantitative trait loci (QTLs). In the past ten years, many QTLs/genes
   regulating grain size have been identified through classical mutant
   analysis or map-based cloning. They are involved in several important
   genetic pathways, mediated by phytohormones, G-proteins, proteasomal
   degradation, protein kinases, and transcriptional factors^[75]4–[76]8.
   Thus far, several genes for grain size, such as GW2, GL3.1, GS5, GW8,
   GS2/GL2, GS3, GL7/GW7 and GS9, have been identified in Asian
   rice^[77]9–[78]15. GS3 was the characterized QTL that regulates grain
   length (GL), encoding a transmembrane protein. The natural variation in
   GS3 contributes to grain-length differences between indica and japonica
   varieties^[79]10,[80]16. GL3.1 is a serine/threonine phosphatase that
   belongs to the PPKL family. It regulates grain length by mediating cell
   cycle progression through affecting the phosphorylation status of cell
   cycle proteins, such as Cyclin-T1;3, thereby controlling grain
   yield^[81]11,[82]12,[83]17. Transcription factor OsMADS1, protein
   phosphatase GL3-1/GL3.1/OsPPKL1 and transcriptional regulator GS9 have
   been reported to control grain length by regulating cell proliferation
   in the grain hull^[84]11,[85]12,[86]17–[87]19. GS2 encodes
   transcription factor OsGRF4, which regulates grain size mainly by
   promoting cell expansion and slightly promoting cell proliferation.
   OsGRF4 is regulated by microRNA396. The GS2 allele with mutations in
   the targeting site of OsmiR396 in OsGRF4 disrupts the repression
   effects of OsmiR396 on OsGRF4, leading to increased expression of
   OsGRF4, and resulting in large grains and high yield. OsGRF4 physically
   interacts with transcription coactivators
   OsGIF1/2/3^[88]5,[89]11,[90]20,[91]21. However, the genetic and
   molecular mechanisms by which these factors control grain size still
   remain to be further elucidated.

   As the direct progenitor of modern rice, common wild rice (Oryza
   rufipogon Griff.) has an important role in cultivated rice breeding and
   been considered as valuable resources for improving rice yield and
   environmental resilience. Cultivated crops, such as wheat, maize, and
   rice, have larger seeds than their wild relatives^[92]22. Almost all
   cultivated rice varieties have larger seeds than rufipogon. Although
   the overall phenotypic characteristics of wild rice, especially
   yield-related traits, are inferior to those of cultivated rice, studies
   have show that genes in wild rice are potentially useful for
   yield-related trait improvement^[93]23. However, so far there are no
   positive regulation alleles for yield reported from wild rice.
   Cultivated rice is also a salt-sensitive plant, wild rice harbors
   especially genes controlling adaptation to unfavorable environments
   which were lost during domestication. The exploration and utilization
   of salt tolerance genes in wild rice resources is the main strategy of
   rice salt tolerance breeding. However, the elite resistant traits often
   linked with inferior yield traits in wild rice. Therefore, the
   dissection of wild rice QTLs/genes that synergistically increase yield
   and salt tolerance would be crucial for rice breeding.

   In our previous study, we identified a QTL-qGL12 from wild rice, which
   regulates grain length in cultivated rice^[94]24. Here, we find that
   the overexpression of the wild rice GL12^W allele simultaneously
   increases grain length and salt tolerance. A G/T variation in the GL12
   promoter region, interacting with transcription factor regulator OsGIF1
   (Growth-regulating factor interacting factor 1) and transcription
   factor OsWRKY53, is involved in the grain length and salt tolerance
   regulating pathways. In addition, it is found that GL12^W functions in
   different rice genetic backgrounds including both indica and japonica.
   Our results provide resources for rice breeding and insights for
   synergistic mechanism of rice yield and salt tolerance.

Results

Identification and confirmation of the wild rice qGL12 candidate gene

   To identify wild rice genes underlying the natural variations in grain
   length (GL) and 1000-grain weight (TGW), we constructed a set of
   chromosome segment substitution lines (CSSLs) with an indica variety
   ‘9311’ as the recurrent parent and the O.rufipogon as the donor parent.
   One major QTL for GL, qGL12, was identified across six environments,
   and mapped using molecular markers on chromosome (Chr.) 12^[95]25. One
   CSSL line, CSSL41, which has a long GL phenotype and carries the
   QTL-qGL12, was selected for further fine mapping (Supplementary
   Fig. [96]1a). An F[2] population was constructed from a cross between
   CSSL41 and 9311 (Fig. [97]1a). Using bulked-segregant analysis (BSA),
   and F[3] and F[4] homozygous recombinant analysis, qGL12 was finally
   narrowed to a~16-kb interval between molecular markers WR12.4 and
   RM28586, which contains three predicted ORFs (Supplementary
   Fig. [98]1b, Fig. [99]1a, b). Among them, LOC_Os12g39640 encodes a MYB
   transcription factor and sequence analysis revealed 11 single
   nucleotide polymorphisms (SNPs) and six insertion-deletion (InDels)
   polymorphism in the 1.8-kb promoter region and four SNPs in the coding
   region causing amino acid change between wild rice and 9311
   (Supplementary Fig. [100]2a). LOC_Os12g39650 encodes a tubulin and
   LOC_Os12g39660 encodes a calcium-transporting ATPase, and there were
   eight and 17 SNPs between wild rice and 9311 in these two alleles
   respectively (Supplementary Fig. [101]3). We designed a cleaved
   amplified polymorphic sequence (CAPS) marker associated with wild rice
   LOC_Os12g39640 allele (Supplementary Fig. [102]2b), and a near-isogenic
   line (NIL) of qGL12 was developed with marker-assisted selection
   (Fig. [103]1a, c). We tested the expression levels of these three genes
   in young panicles of the NIL and 9311, among them, expression levels of
   LOC_Os12g39640 and LOC_Os12g39650 were significantly higher in the NIL
   than in 9311 (Fig. [104]1d). Compare with 9311, the NIL showed
   significantly increased GL and TGW, with 8.1% and 12.9%, respectively
   (Fig. [105]1f, g). To identify the candidate gene, we overexpressed
   (OE) these three genes from wild rice in 9311 background individually,
   both of LOC_Os12g39640-OE and LOC_Os12g39650-OE showed significantly
   increased GL (5.1% and 2.2%, respectively) than 9311, and
   LOC_Os12g39640-OE have much more increased GL than that of
   LOC_Os12g39650-OE (Fig. [106]1e–g). Given LOC_Os12g39650 is a tubulin
   gene, the LOC_Os12g39640 gene is the most likely candidate gene.
   Moreover, we knocked out (KO) LOC_Os12g39640 in the NIL background
   using CRISPR/Cas9 technology. The GL and TGW of NIL-KO plants decreased
   significantly, and the overexpression of LOC_Os12g39640 from 9311 did
   not improve GL in 9311 genetic background^[107]24 (Supplementary
   Fig. [108]4), which confirmed that LOC_Os12g39640 was the candidate
   gene and therefore termed GL12.

Fig. 1. Wild rice GL12^W increases the grain length and weight in different
genetic background.

   [109]Fig. 1
   [110]Open in a new tab

   a The near isogenic line (NIL) of qGL12 and recurrent parent 9311. b
   Fine mapping of qGL12. c Schematic diagram showing 9311 and wild rice
   chromatin. d Expression analyses of three genes in the qGL12 interval.
   The 2–5 cm young panicles were used for RT-qPCR. The relative
   abundances in 9311 plants were set to be one. Values given are
   means ± SD (n = 3) Statistical analysis was performed by two-tailed
   Student’s t-test *p ≤ 0.05, **p ≤ 0.01, NS no significant difference.
   The p-values were 3.7 × 10^–4,0.028 and 0.086, respectively. e Grains
   of 9311, NIL, and overexpression lines of three genes in qGL12.
   Bar = 1 cm. f, g Statistical analysis of grain length (GL) and
   1000-grain weight (TGW) of lines in (e). Data were given as means ± SD
   (n = 21/20/22/21/19, 21/20/22/21/19 biological replicates). One-tailed
   Student’s t-test was used to generate the P values. h Grains of GL12
   overexpression lines in Zhonghua11(ZH11) genetic background.
   Bar = 1 cm. i, j Statistical analysis of grain length and 1000-grain
   weight of lines in (h). Data were given as means ± SD
   (n = 21/20/22/21/19, 21/20/22/21/19 biological replicates). One-tailed
   Student’s t-test was used to generate the P values. Source data are
   provided as a Source Data file.

   In addition, we generated transgenic lines in japonica variety
   ‘Zhonghua11 (ZH11)’ background, which overexpressed GL12/LOC_Os12g39640
   alleles from wild rice (GL12^W) and 9311 (GL12^9311), respectively. The
   GL12^W-OE lines showed significantly increase in GL and TGW than wild
   type ZH11 while GL12^9311-OE lines had no difference (Fig. [111]1f–j,
   Supplementary Fig. [112]5). We also overexpressed GL12^W in ‘Nipponbare
   (NIP)’ background and the OE lines showed significantly increase in GL
   and TGW than that of wild type (Supplementary Fig. [113]6d, e). These
   data demonstrated that GL12^W has stable positive effect on GL in
   different genetic backgrounds.

GL12^W improves grain length by reprograming genes expression and altering
cell length

   GL12 encodes a MYB transcription factor, which belongs to a family with
   more than 2,00 members in rice^[114]26,[115]27. From the NCBI database,
   we performed phylogenetic analysis and revealed that GL12 was clustered
   with MYB transcription factor gene family in rice and other species,
   closest with japonica homologous gene (Supplementary Fig. [116]7). The
   GL12^w::GFP fusion protein was localized in the nucleus in tobacco
   epidermal cells and rice protoplasts (Fig. [117]2a). These results
   indicated that the GL12 protein could function in the nucleus and was
   consistent with the role of GL12 as a transcription factor.

Fig. 2. GL12^W regulated grain size related genes and improves grain length
by altering cell length.

   [118]Fig. 2
   [119]Open in a new tab

   a Subcellular localization of GL12 (35S::GL12-GFP) in tobacco leaf
   cells and rice protoplasts. The fusion construct 35S::GFP/RFP marker,
   and GL12 (35S::GL12-GFP) / RFP marker were co-transformed into rice
   protoplasts and tobacco leaf cells, respectively. Scale bar = 10 μm. b
   Heat map diagram of the expression levels for rice seed size related
   genes. Data were from transcriptome using 2–5 cm young panicles. The
   heat map was drawn according to FPKM values. Columns and rows in the
   heat map represent samples and genes, respectively. Color scale
   indicates fold changes in gene expression. c Transcription levels of
   the grain size related genes were confirmed through qRT-PCR analysis
   with three biological replicates in 9311 and NIL. OsActin was used as a
   control. Data show means ± SD. d–f Spikelet hulls before anthesis (d).
   The white dashed line indicates the sites of the cross-sections shown
   in (e). The images on the right panel showed the close-up views of the
   boxed region in (e), Scale bar = 100 μm. The red arrows indicate the
   lower epidermal cells. Comparisons of spikelet of cell number, and cell
   length between 9311, NIL and GL12^W transgenic plants were shown in
   (f), letters a–c in indicate the different means according to Duncan’s
   multiple range test (P < 0.05). g Quantitative real-time PCR analysis
   of cell cycle-related and expansion gene expression in 9311, NIL and
   GL12^W-OE lines. OsActin was used as a control. Data show means ± SD of
   three biological replicates. Statistical analysis was performed by
   two-tailed Student’s t-test (*p ≤ 0.05, **p ≤ 0.01, NS no significant
   difference). Scanning electron microscopic analysis of the lemma of NIP
   and GL12^W-OE lines (h), and statistical analysis of longitudinal cell
   number and cell length in the lemmas of spikelet hulls (i) n = 10 view
   of 10 areas in (i). Statistical analysis was performed by two-tailed
   Student’s t-test (***p < 0.001). Source data are provided as a Source
   Data file.

   To identify the molecular mechanism and downstream genes regulated by
   GL12^W, we first performed RNA-sequencing analysis using young panicles
   from NIL and NIL-KO plants. As a result, 8079 differentially expressed
   genes (DEGs) were identified with the stringent criteria (|log 2
   (FC)|> 1, and FDR < 0.05). Among these DEGs, 4615 were upregulated and
   3464 were downregulated in the NIL compared with the NIL-KO lines. Gene
   Ontology identified that GL12^W mostly participated in biological
   processes, and KEGG pathway enrichment analysis revealed that GL12^W
   affected the expression of genes involved in metabolism, genetic
   information processing, environmental information processing,
   organismal systems, and cellular processes (Supplementary Fig. [120]8).
   In addition, we found that several grain shape related genes were
   downregulated in young panicles of the NIL-KO relative to the NIL
   (Fig. [121]2b), including previously reported GS2/GRF4 genes^[122]11.
   We also confirmed the expression levels of grain shape related genes in
   the NIL and 9311, GS2, GRF3, and GRF6 had significant higher expression
   levels in the NIL than that of 9311 (Fig. [123]2c). These results
   indicated that GL12^W might regulate grain shape by affecting the
   expression of GRFs.

   The rice spikelet hull, which is coordinately controlled by cell
   proliferation and cell expansion, was proposed to restrict the rice
   grain shape^[124]4. To further understand the function of GL12 in grain
   shape, we investigated the spikelet hulls of 9311, GL12^W-OE and NIL
   before fertilization. The results verified that the longer GL in the
   NIL and GL12^W-OE was the result of cell expansion in spikelet hull
   (Fig. [125]2d, e). Furthermore, we examined the cross-section of
   spikelet hulls, and showed that the outer parenchyma cell layer in
   GL12^W-OE and NIL plants were longer and had substantially longer
   spikelet perimeter than the corresponding layer in 9311 plants
   (Fig. [126]2f). Cell division and expansion in eukaryotic organisms are
   controlled by a highly conserved basic cell cycle
   machinery^[127]28,[128]29. To investigate the relationship of GL12 with
   genes regulating plant cell cycle and expansion, we analyzed the
   expression of eight related genes in the NIL and GL12^W-OE relative to
   9311 by real-time PCR. The transcription levels of five genes, CYCD4,
   CYCD7, CAK1, CYCA2.1 and CYCB2.2, were greatly reduced in GL12^W-OE and
   NIL as compared to 9311 plants. In contrast, the expression of OsEXPB3,
   OsEXPB4 and OsEXPA10 were significantly elevated in the NIL and
   GL12^W-OE (Fig. [129]2g). Additionally, we checked the grain hulls of
   GL12^W-OE NIP lines and NIP wild type using a scanning electron
   microscope. There were significantly fewer longitudinal cells in
   spikelet hulls of GL12^W-OE than in NIP; however, the cells of spikelet
   hulls of GL12^W-OE were significantly longer than those of Nip
   (Fig. [130]2h, i). Thus, GL12^W functions putatively as a positive
   regulator upstream of cell expansion genes, and its overexpression may
   result in an increase in cell expansion in longitudinal direction.
   These findings suggested that GL12^W functions as a regulator of both
   cell proliferation and cell expansion.

The native promoter enhances GL12^W expression in young panicle and increases
grain length

   Compared with GL12^W, GL12^9311 coding region has four mutant sites and
   has no function on GL improvement (Supplementary Fig. [131]2a). There
   were 17 SNPs/InDels in the promoters of GL12 between 9311 and wild rice
   (Supplementary Fig. [132]2a). We performed real-time PCR to detect the
   expression of GL12 in young panicles of 9311 and the NIL, and found
   that the transcription levels of GL12 in the NIL were significantly
   higher in all panicle development stages than those in 9311
   (Fig. [133]3a). We then performed the transient expression assays in
   Nicotiana benthamiana leaves, and found that the promoter of GL12^W
   (Pro^W) had stronger activity than that of GL12^9311 (Pro^9311)
   (Fig. [134]3b, c). Furthermore, we generated transgenic plants in the
   NIP background that harbored the promoter and coding regions of GL12
   from 9311 and NIL, respectively. We found that the GL of Pro^W:GL12^W
   transgenic lines were much longer than those of NIP and the
   Pro^9311:GL12^W transgenic lines and NIP (4.4% and 3.5% increase than
   NIP and Pro^9311:GL12^W, respectively) (Fig. [135]3d–f), demonstrating
   that GL12^W has a stronger effect on GL under its native promoter.

Fig. 3. Promoter activity assays for the GL12 alleles in 9311 and wild rice.

   [136]Fig. 3
   [137]Open in a new tab

   a Relative quantitative real-time PCR analysis of GL12 expression in
   young panicles of 2, 4, 6, 7, and 8 cm in 9311 and NIL. OsActin was
   used as a control. qRT-PCR analysis with three biological replicates.
   Data showed means ± SD. (The p-values were 2.9 × 10^–5, 3.5 × 10^–5,
   2.7 × 10^–3,1.7 × 10^–3and 3.4 × 10^–6, respectively). b Transient
   expression assays in Nicotiana benthamiana leaves showing the promoter
   activities of GL12, and the quantification of the relative luminescence
   intensities were shown in (c) The quantification of the relative
   luminescence intensities (Data show means ± SD, n = 8). Statistical
   analysis was performed by one-tailed Student’s t-test. d Grains of the
   transgenic lines in Nipponbare background. GL12 alleles form the wild
   rice and 9311 were driven by both promoters. Bar = 1 cm. Statistical
   analysis of grain length (e) and grain weight (f) in transgenic plants
   (n = 30/31/31/31/29). Data were given as means ± SD. One-tailed
   Student’s t-test was used to generate the P values. Source data are
   provided as a Source Data file.

The G/T variation in GL12 promotes its binding by GIF1

   The OsGIF1 (GRF-interacting factor) gene in rice, which encodes a
   transcription coactivator, has been reported to interact with the
   transcription factor GS2/OsGRF4 and positively promote grain size in
   rice^[138]30–[139]32. Firstly, we detected the physical interaction
   between GL12 and OsGIF1. The yeast two-hybrid assay (Y2H) and firefly
   luciferase complementation experiment showed that both GL12^W and
   GL12^9311 can interact with OsGIF1 protein (Fig. [140]4a, b). To
   determine whether GL12 was regulated by GIF1, we investigated the
   expression levels of GL12 in the GIF1 overexpression and CRISPR/Cas9
   knock out lines in ZH11 background. Compared to ZH11, the GL12
   expression levels in GIF1-OE lines were significantly improved while no
   significantly difference was found in GIF1-KO lines (Fig. [141]4c).
   Furthermore, we generated four constructs with truncated promoters of
   Pro^W and Pro^9311 of GL12 alleles from NIL and 9311, respectively
   (Fig. [142]4d). The yeast one-hybrid assay results demonstrated that
   GIF1 could only bind the promoter of Pro-2, and GIF1 bind the Pro^W-2
   much stronger than Pro^9311-2. (Fig. [143]4d; Supplementary
   Fig. [144]9). Unexpectedly, the truncation of Pro^W-2 only harbored one
   SNP between the wild rice and 9311, which generated a G/T substitution
   at the -1117th position (Fig. [145]4d). These results were further
   supported by the electrophoresis mobility shift assay (EMSA) that used
   biotin-labeled probes containing the G/T SNP in the Pro^W-2
   (Fig. [146]4e). These results indicated that the G/T site contributed
   to the binding of GIF1 to the promoter of GL12 and that was a direct
   target regulated by GIF1. Moreover, we knocked out the OsGIF1 in NIL
   background by CRISPR/Cas9 technology, the grain length of GIF1-KO-NIL
   significantly decreased (Supplementary Fig. [147]10).

Fig. 4. The G/T variation in GL12 promotes its binding by GIF1.

   [148]Fig. 4
   [149]Open in a new tab

   a Yeast two-hybrid assay showed that GIF1 physically interacted with
   GL12^W and GL12^9311. b LCI assays in N. benthamiana leaves showing the
   GIF1 and GL12 interaction. N. benthamiana leaves were transformed by
   injection of Agrobacterium GV3101 cells harboring GL12-nLUC and
   GIF1-cLUC plasmids. Luciferase complementation was observed for the
   GL12-nLUC and cLUC- GIF1 combination, the negative control was
   GL12-nLUC + NAC2-cLUC, NAC2 was a nuclear localization gene, whereas no
   obvious signal was observed for the negative controls. c The GL12
   expression levels in ZH11 and GIF1 overexpression (GIF1-OE) and
   knockout (GIF1-KO) lines. OsActin was used as a control. qRT-PCR
   analysis with three biological replicates. Statistical analysis was
   performed by two-tailed Student’s t-test (*p ≤ 0.05, **p ≤ 0.01, NS no
   significant difference). Data showed means ± SD. d Schematic
   illustration of the four truncations of GL12 promoters. The MYB motif
   was highlighted in yellow and the G/T variation was marked in red.
   Yeast one-hybrid assays were shown. GIF1 only interacted with Pro^W-2
   truncation. Other yeast one-hybrid results were shown in Supplementary
   Fig. [150]9. e EMSA assays showed that GIF1 bound to the Pro^W-2
   truncation. EMSA assays were repeated three times. GST-GIF1 fusion
   protein was expressed in E. coli. The probes were labeled with biotin.
   Source data are provided as a Source Data file.

GL12^W improves salt tolerance in rice

   Besides GL12^W could increase the grain length and grain weight, we
   found that GL12^W could also improve salt tolerance. After extensive
   salt treatment (10 days of 150 mM NaCl treatment, recovered for seven
   days), both the NIL and GL12^W-OE showed significant higher salt
   tolerance than that of 9311, and the NIL showed much stronger salt
   tolerance than GL12^W-OE lines. The survival rate of the NIL, GL12^W-OE
   lines, and 9311 were 80%, 60% and no more than 30%, respectively. And
   the fresh weight and dry weight of the NIL were significantly higher
   than that of 9311 and GL12^W-OE lines. Meanwhile the NIL-KO line showed
   hypersensitivity to salt stress (Fig. [151]5a, b). Moreover, the
   overexpression of GL12^W in the NIP genetic background also
   significantly improved salt tolerance, compared with NIP wild type
   plants (Supplementary Fig. [152]11). There was no significant
   difference in salt tolerance between GL12^9311-OE lines and 9311
   (Supplementary Fig. [153]12). We then investigated the GL12 expression
   pattern under salt treatment. Before salt treatments, GL12 had higher
   basic expression levels in leaves of 9311 than that of NIL; after salt
   treatment, the GL12 expression in 9311 was induced rapidly compared
   with that in the NIL (Fig. [154]5c). These results indicated that
   GL12^W and GL12^9311 have different expression patterns under salt
   treatment. Moreover, we analyzed the expressions of salt
   tolerance-related genes in the NIL and 9311. Compared with 9311, the
   NCED3 and NAC5 in the NIL were significantly induced after salt
   treatments (Fig. [155]5d, e). We further determined the effects on salt
   tolerance of OsGIF1 and GS2, and found that the GIF1-OE lines showed
   higher salt tolerance while GIF1-KO lines were salt sensitive compared
   with wild type ZH11 (Supplementary Fig. [156]13). The GS2-NIL line,
   which had increased GS2 expression levels^[157]11, also showed higher
   salt tolerance than its genetic background ZH11 (Supplementary
   Fig. [158]13). These results indicated that GL12^W also played an
   important role in regulating the salt stress response in rice.

Fig. 5. GL12^W improves salt tolerance in rice.

   [159]Fig. 5
   [160]Open in a new tab

   a Photos of 9311, NIL and GL12^W overexpression lines before and after
   salt treatments. 20-day-old seedlings were treated with 150 mM NaCl
   for10 days and recovered for seven days. b Statistical analysis of
   survival rates and fresh and dry weight of seedlings in (a). Five
   biological replicates were used to determine the survival rate. Data
   were means ± SD (n = 5). Statistical analysis was performed by
   two-tailed Student’s t-test (*p ≤ 0.05, **p ≤ 0.01, NS no significant
   difference). c GL12 expression analysis in 9311 and NIL. 20-day-old
   seedlings were treated with 150 mM NaCl for indicated time. qRT-PCR
   analysis with three biological replicates. Data were means ± SD.
   Expression analysis of rice salt tolerance related genes NAC5 (d) and
   NCED3 (e) in 9311 and NIL, 20-day-old seedlings were treated with
   150 mM NaCl. OsActin was used as a control. qRT-PCR analysis with three
   biological replicates. Data showed means ± SD. Source data are provided
   as a Source Data file.

The G/T variation in GL12 promoter determines its repression by WRKY53

   Compared with GL12^9311, the GL12^W expression was higher in young
   panicles but lower in leaves under salt stress (Figs. [161]3a,
   [162]5c). We postulated that GL12^W might be regulated by another
   transcription factor. Because OsWRKY53 was reported to be a key
   regulator of salt tolerance in rice^[163]33, we investigated whether
   GL12 was involved in the OsWRKY53 regulated salt tolerance pathway. The
   OsWRKY53-OE and CRISPR/Cas9 knockout lines in japonica variety
   ‘LongGeng 11’ (LG11) background were used to determine the GL12
   expression levels. The GL12 expression was significantly increased in
   the WRKY53-KO lines and significantly decreased in the WRKY53-OE lines,
   compared with wild type LG11 (Fig. [164]6a), which was consistent with
   the WRKY53-KO lines showed stronger salt tolerance^[165]33. To study
   the interaction between WRKY53 and GL12, we analyzed the promoter
   region of Pro-1 to Pro-4 of GL12^W and GL12^931. Consistent with GIF1,
   the Y1H experiments demonstrated that WRKY53 could only bind the
   truncated promoter Pro-2 (Fig. [166]6b; Supplementary Fig. [167]9),
   which harbored only one G/T variation between the wild rice and 9311
   (Fig. [168]4d). Interestingly, Y1H and EMSA proved that the binding
   ability of Pro^W-2 with WRKY53 was stronger than that of Pro^9311-2
   (Fig. [169]6c). We further tested the capacity of WRKY53 protein on
   driving the expression of GL12 using dual-luciferase assays. The
   promoter of GL12 was fused to the luciferase reporter gene and
   co-introduced into rice protoplast with constructs driving the
   expression of WRKY53 (Fig. [170]6d). In the presence of WRKY53, the
   luciferase (LUC) activity driven by the promoter of GL12^W was largely
   reduced than that of GL12^9311 (Fig. [171]6e, f). These results
   strongly suggested that WRKY53 regulated salt tolerance by repressing
   the GL12^W expression.

Fig. 6. The G/T variation in GL12 determines its repression by WRKY53.

   [172]Fig. 6
   [173]Open in a new tab

   a The GL12 expression levels in Longgeng11 (LG11) wild type, and WRKY53
   overexpression and knockout lines. Young leaves from 20-day-old
   seedlings were used for RNA extraction and Real-time PCR analysis.
   OsActin was used as a control. qRT-PCR analysis with three biological
   replicates. Data showed means ± SD. Statistical analysis was performed
   by two-tailed Student’s t-test (*p ≤ 0.05, **p ≤ 0.01, NS no
   significant difference). b Yeast one-hybrid assays proved that the
   transcription factor WRKY53 only bound to the Pro-2. Other yeast
   one-hybrid assay results were shown in Supplementary Fig. [174]9. c
   EMAS assays showed that WRKY53 bound to Pro^W-2. EMSA assays were
   repeated three times. MBP-WRKY53 fusion protein was expressed in E.
   coli. The probes were labeled with biotin. d Schematic diagrams of the
   effectors and reporters used in the dual-LUC experiments. Transient
   expression assays in N. benthamiana protoplasts showing that WRKY53
   could reduce the transcriptional activity of GL12^W (f), but not the
   GL12^9311 (e). The activities of firefly luciferase (LUC) and renilla
   luciferase (REN) were determined 16 h post transformation. The relative
   luciferase activities in different samples were calculated by
   normalizing the LUC values against REN. LUC/REN analysis with five
   biological replicates. Statistical analysis was performed by two-tailed
   Student’s t-test (*p ≤ 0.05, **p ≤ 0.01; NS, not significant). Data
   show means ± SD. Source data are provided as a Source Data file.

Natural variations in GL12 contribute to the divergence of indica and
japonica subspecies

   To identify whether GL12 has been artificially selected during rice
   domestication, we further analyzed the genomic sequences of this gene
   in rice germplasm resources from the 3K Rice Genomes
   Project^[175]34,[176]35, the Rice Super Pangenome Information Resource
   Database (RiceSuperPIRdb)^[177]36 and our own re-sequencing data.
   Firstly, we compared the nucleotide diversity (PI-value) of the 100-kb
   region flanking GL12 with our re-sequencing data (56 indica, 51
   japonica, and 52 wild rice accessions). The results showed that the PI
   values of the GL12 locus in wild rice were significantly higher than
   those in indica cultivars, but no significant difference with those
   japonica cultivars. In addition, the PI ratio in indica to that in wild
   rice was significantly lower than in japonica to that in wild rice in
   the GL12 locus (Fig. [178]7a, b).

Fig. 7. Natural variations in GL12 contribute to the divergence of indica and
japonica subspecies.

   [179]Fig. 7
   [180]Open in a new tab

   a, b The relative ratio of nucleotide diversity analysis in the
   complete chromosome 12 of cultivated and common wild rice. The GL12
   locus and its flanking genomic regions were marked. Dotted line area
   indicates a selective sweep of GL12 locus in indica. c Distribution of
   grain length for the two haplotypes in indica rice accessions,
   n = 15/894 means the accessions number of HapG and HapT. The haplotypes
   and number of accessions from the 3K Rice Genomes Project dataset were
   shown. Data show means ± SD. Statistical analysis was performed by
   two-tailed Student’s t-test (*p ≤ 0.05, p-value was 0.0104). d
   Haplotype networks generated from the promoter sequence of GL12 in both
   cultivated rice and various groups of wild rice accessions. The circle
   size is proportional to the sample size. Grey spots on the lines
   indicate the mutational steps between two haplotypes. e A proposed
   working model for the role of GL12 in the regulation of rice grain
   length and salt tolerance. Wild rice GL12^W is a functional and
   GL12^9311 is a nonfunctional allele. The G/T variation in GL12 promoter
   determined its binding with transcription coactivator GIF1 and
   transcription factor WRKY53. GIF1 promotes GL12^W expression levels in
   young panicles, and then regulates the downstream grain size related
   genes, such as GS2. WRKY53 represses GL12^W expression under salt
   stresses, and GL12^W also regulates rice salt response genes, such as
   NCED3 and NAC5. Therefore, the wild rice allele GL12^W synergistically
   improves both the rice grain length and salt tolerance through the
   regulation by GIF1 and WRKY53. The image was drawn by Figdraw. Source
   data are provided as a Source Data file.

   Furthermore, with the data from the 3K Rice Genome Project and rice
   super pangenome data, we found that the G/T substitution (1117-bp
   upstream of the start codon) in GL12 was unbalanced between the two
   cultivated rice subspecies, the majority of indica varieties were
   haplotype-T at the G/T site whereas most of japonica varieties were
   haplotype-G (Supplementary Fig. [181]14). We further investigated the
   role of G/T variation in the promoter of GL12 on GL using the data from
   3K Rice Genomes Project of the indica varieties. Among them, 15 and 894
   indica varieties carried the G and T types, respectively. The indica
   varieties with the haplotype-G exhibited longer grains than those with
   the haplotype-T (Fig. [182]7c). The haplotype classification results
   showed that the 228 rice varieties could be divided into a total of 20
   haplotypes (Fig. [183]7d). Interestingly, the rice varieties within the
   blue dashed line are all Hap-T at promoter -1117 position, while those
   within the red dashed line are Hap-G. Among them, most japonica are
   Hap-G, indica are Hap-T, and wild rice are distributed in both
   haplotypes. And the Aus rice tends towards the indica type. All these
   findings suggested the genetic differentiation of GL12 between japonica
   and indica subspecies, which exhibited significantly reduced nucleotide
   diversity in indica compared with wild rice, suggesting a strong
   artificial selection in the GL12 locus of indica.

   We proposed a possible working model for the network and showed that
   GL12 played an essential role in grain length and salt tolerance, which
   was synergistically regulated by GIF1 and WRKY53 at the G/T variation
   site (Fig. [184]7e).

Discussion

   Early attempts to identify positive yield-related genes proved
   unsuccessful because most of the agronomic traits, especially
   yield-related characters of wild rice were inferior to those of
   cultivated rice. In this study, we identified wild rice gene GL12^W
   that improved rice GL and salt tolerance, which is synergistically
   regulated by GIF1 and WRKY53. The wild rice CSSL population we
   development in our previous study has greatly facilitated the discovery
   of unknown useful genes in wild rice^[185]37. We identified the GL12^W
   gene using CSSL population and found it improves both the GL and salt
   tolerance synergistically in cultivated rice.

   Compared with GL12^9311, GL12^W improved the GL, TGW and salt tolerance
   in both indica and japonica genetic backgrounds (Figs. [186]1, [187]5;
   Supplementary Figs. [188]6, [189]9). We demonstrated that GL12^W
   regulated global gene expression, including GS2, and improves the GL by
   altering cell length (Fig. [190]2). We found a G/T variation at
   1,117-bp upstream of the start codon of GL12 playing an essential role
   in binding with OsGIF1 and OsWRKY53, which positively and negatively
   regulated the GL12 expression, respectively (Figs. [191]3, [192]6).
   Previous reports demonstrated that OsGIF1 interacted with GS2 to
   control grain-size predominantly by increasing cell expansion in the
   grain hull^[193]11,[194]20,[195]30. But whether these two genes
   involving in rice salt tolerance remains unknown, and no reports on
   whether GIF1 interacted with DNA and regulates gene expression
   directly. Our results showed the GIF1 physically interacted with GL12
   but no differences were found between GL12^W and GL12^9311
   (Fig. [196]4a, b). GIF1 interacted with GL12^W promoter and the G/T
   site determined its binding to GIF1 and increased the GL12^W expression
   levels in young panicle (Fig. [197]4c–e). We also found that GIF1 and
   GS2 positively regulated salt tolerance in rice (Supplementary
   Fig. [198]13). Our results provided insights into the GIF1-GL12^W-GS2
   module how to regulate the grain length and salt tolerance.
   Nevertheless, further studies are needed to explore the detailed
   mechanism and regulatory network of this module.

   The NIL of GL12^W showed longer GL and stronger salt tolerance than
   GL12^W-OE lines. In addition, our transgenic experiments demonstrated
   that GL12^W had a stronger effect on the GL under its native promoter
   (Fig. [199]3d–f). Interestingly, compared with GL12^9311, GL12^W has
   higher expression levels in young panicle but lower expression levels
   under salt stresses, and the latter might be caused by the repression
   of WRKY53 (Fig. [200]6). The expression levels of GL12^W
   synergistically regulated by GIF1 and WRKY53, reach an optimal balance
   in NIL, that explains NIL had much longer grain length and higher salt
   tolerance than GL12^W-OE lines. We further demonstrated that the G/T
   variation contributed to the divergence of indica and japonica
   subspecies. GL12 might have been directionally selected in indica but
   not in japonica during rice domestication (Fig. [201]7d). We
   investigated the agronomic and yield-related traits of 9311, NIL and
   GL12^W-OE lines, GL12^W has improved plant height, panicle length, but
   significantly decreased the grain number per panicle, same results were
   found in GL12^W-OE lines in NIP background (Supplementary
   Fig. [202]15). Unfortunately, the GL12^W did not improve yield
   significantly under both normal and salt stress conditions. This
   indicated that GL12^W might have a negatively effect on the grain
   number per spike, which can explain why the wild rice GL12^W has not
   been selected from wild rice to indica varieties during the rice
   artificial selection breeding.

   Improving the salt tolerance without reducing yield is one of the major
   goals for modern rice breeding. The wild rice GL12^W gene, regulated by
   GIF1 and WRKY53, provides the endogenous cues for wild rice gene
   utilization and further paves the way for future investigation of the
   detailed biochemical mechanisms that synergistically improve both the
   grain length and salt tolerance. In summary, the discovery of this gene
   not only helps understand the mechanism for improving grain length and
   environmental resilience simultaneously for rice improvement but also
   provides the useful genetic resources for rice breeding programs.

Methods

Plant materials and growth conditions

   Rice (O. sativa L.) materials include two wild-type japonica rice
   varieties, ZH11 and NIP, and one indica rice 9311. The overexpression
   lines were derived from ZH11 and NIP, and the knockout mutants were
   from NIL. All rice plants were cultivated in a natural paddy condition
   at Beijing and Sanya in Hainan province, China. Standard cultivation
   practices were used, and pests and diseases were managed in rice
   cultivation. Mature and dry seeds were used for measuring grain length,
   width, and TGW by image analysis method using SC-E software (Hangzhou
   Wanshen Detection Technology, Hangzhou, China). The plant height,
   panicle length, tiller number, and grain number were obtained from the
   measurement of the main culm. All agronomic traits and grain size
   measurements were based on at least 20 individuals, which were randomly
   selected.

Vector construction and rice transformation

   To construct the overexpression vector, the full-length coding sequence
   of GL12 was amplified from the wild rice and 9311 and cloned into a
   pBWA(V)HS vector. Genes were under the control of double 35S promoter
   in overexpression lines. The Pro^9311::GL12^9311, Pro^9311::GL12^W,
   Pro^W::GL12^9311 and Pro^W::GL12^W transgenic lines also used the
   pBWA(V)HS vector, but 35S promoter fragment of the pBWA(V)HS vector
   were replaced by the GL12 promoter from the wild rice or 9311
   respectively, the construct vectors were introduced into the calli of
   NIP and by Agrobacterium mediated transformation. To generate knockout
   mutants, two GL12 site-specific target sequences were selected in the
   fifth exon and introduced into the CRISPR/Cas9 system, and the plasmid
   were transformed into GL12^W-NIL to generate the GL12 knockout mutants.
   Knockout lines of OsGIF1 in the GL12^W-NIL background were also
   generated by CRISPR/Cas9 technology.

RNA extraction and RT-PCR

   Total RNA was extracted from young leaves and panicles using the RNA
   Easy Fast Plant Tissue Kit (TIANGEN, Beijing, China) and
   reverse-transcribed into the first-strand cDNA with a PrimeScript RT
   Reagent Kit (Invitrogen, Carlsbad, CA). Real-time qPCR experiments were
   performed using a SuperReal PreMix Plus (SYBR Green) (TIANGEN) on an
   Applied Biosystems 7500 Real-Time PCR system following the
   manufacturer’s instructions. All the qRT-PCR analyses were performed on
   at least three independent biological replicates. Rice ACTIN was used
   as an internal control for all the qRT-PCR analyses. Primers for
   qRT-PCR are listed in Supplementary Data [203]1.

Subcellular localization

   To generate the 35S::GL12-GFP fusion, the full length cDNA of GL12
   without the stop codon was cloned into vector pCAMBIA 1300. The empty
   35S: GFP, 35S::GL12-GFP and NLS-RFP plasmids were transformed into
   Agrobacterium tumefaciens strain GV3101, and infiltrated into
   3‐week‐old N. benthamiana leaves. The Agrobacteria harboring the
   corresponding construct and NLS-RFP were co-infiltrated into N.
   benthamiana leaves. At 2–4 day after infiltration, fluorescent signals
   in leaves were detected by laser confocal microscopy (Carl Zeiss,
   LSM780, Germany).

Histological analysis

   The fresh young spikelet hulls were fixed using FAA (40% formaldehyde:
   50% alcohol: 5% acetic acid) and embedded in Paraplast Plus (Sigma,
   Germany) then cut into 10-μm-thick sections. Cross sections were
   observed by light microscopy (Olympus, Japan), and the cell number and
   cell area in the outer parenchyma cell layer of hulls were measured
   using ImageJ software^[204]38. The outer surfaces of the spikelet hulls
   were observed using a scanning electron microscope (GeminiSEM 300, Carl
   Zeiss, Germany). Cell size and cell number were measured with ImageJ
   software.

Dual-Luciferase transcriptional activity assay

   To construct the Pro^9311::LUC and Pro^W::LUC plasmids, we amplified a
   2 kb promoter sequence from 9311 and wild rice and cloned them into the
   pGreen0800II-LUC vector, respectively; the resulting vectors were used
   as reporters. The full-length cDNA of WRKY53 and GIF1 were cloned into
   the pGreen0800II-62-SK vector to construct 35S: WRKY53 and 35S: GIF1,
   which were used as the effector. Transient transactivation assays were
   performed using the tobacco (N. benthamiana) leaf system. The LUC and
   REN activities were measured by the Dual-Luciferase Reporter Assay
   System (Promega, USA), and the LUC activity was normalized to REN
   activity. Primers used for these constructs are listed in Supplementary
   Data [205]1.

Yeast one-hybrid assay

   The full-length cDNA of WRKY53 and GIF1 were cloned into the pB42AD
   vector to generate prey constructs. Approximately 2 kb of the GL12
   promoter from wild rice and 9311 were cloned into the lacZ
   (β-galactosidase) reporter plasmid pLACZi as bait constructs. Yeast
   strain EGY48 was used for transformation. All the procedures were
   performed according to the manufacturer’s protocol (Clontech, USA).

Luciferase complementation imaging (LCI) Assay

   The LCI assays for the interaction between GL12 and GIF1 were performed
   in N. benthamiana leaves. The full-length GL12 and GIF1 coding regions
   were fused with the N-terminal and C-terminal parts of the luciferase
   reporter gene, respectively. The nLUC-GL12 and cLUC-GIF1 constructs
   were transformed into Agrobacterium GV3101 cells and co-infiltrated
   into N. benthamiana leaves. One same subcellular localization gene,
   NAC2, was used as the negative control^[206]39,[207]40. The LUC
   activity was analyzed using Tanon 5200 plant imaging system (Tanon
   Science & Technology, China).

Electrophoretic mobility shift assay

   Electrophoretic mobility shift assay (EMSA) was used to examine the
   binding of the WRKY53 and GIF1 proteins to the motif in the promoter
   regions of Pro^9311 and Pro^W, respectively. We synthesized 5′- biotin
   labeled DNA fragments containing binding sites as probes and used
   unlabeled DNA fragments of the same sequence for competitive reactions.
   The MBP-OsWRKY53 and GST-GIF1 fusion protein were expressed in E. coli
   pMal-c2x at 16 °C for 20–24 h in the presence of 0.1 mM isopropyl
   β-D-1-thiogalactopyranoside. The LightShift™ Chemiluminescent EMSA Kit
   was used to perform EMSA according to the manufacturer’s instructions
   (Thermo Fisher Scientific, USA). The binding reactions were incubated
   for 30 min at room temperature and then resolved by electrophoresis on
   6% (w/v) native polyacrylamide gels in 0.5×TBE buffer. Probe sequences
   are shown in Supplementary Data [208]1.

Yeast two-hybrid assay

   Matchmaker Gold Yeast Two-Hybrid System (Clontech, USA) was used for
   Y2H assays. The CDS of GL12 was cloned in prey vector pGBKT7, and the
   GIF1 were cloned into the bait vector pGADT7. The prey and bait vectors
   were transformed into the Y2H Gold yeast. All the procedures were
   performed according to the manufacturer’s protocol (Clontech, USA).
   Relevant primers are listed in Supplementary Data [209]1.

Haplotype and evolutionary analyses

   For the Hap-G and Hap-T distribution analysis, 1772 (596 japonica, 1176
   indica) and 909 (79 japonica, 191 indica, 16 rifipogon) accessions from
   the 3K Rice Genomes Project and rice pan-genome database^[210]36,
   respectively, were used (Supplementary Data [211]2 and [212]3). The
   nucleotide diversity (π) of 52 wild rice, 56 indica, and 51 japonica
   accessions were calculated in 50-kb sliding windows using
   VCFtools^[213]41 and the ratio of genetic diversity in all accessions
   was calculated. The genomic sequences and grain length of 909
   cultivated indica rice were obtained from the 3K Rice Genomes Project
   database. The grain length of two types of promoters of GL12 with T or
   G haplotype was compared using t-test. The geographical distribution of
   909 cultivated rice were obtained from the 3K Rice Genomes Project
   database and marked on map using R-version (Team, 2014). The genotype
   of 219 accessions including wild rice were from rice pan-genome
   database^[214]36. Fifteen SNPs and five InDels of GL12 in the promoter
   were aligned with ClustalW. Haplotype frequency data were processed
   with DnaSP5^[215]42 and visualized Median-joining networks were
   generated by Popart^[216]43.

Reporting summary

   Further information on research design is available in the [217]Nature
   Portfolio Reporting Summary linked to this article.

Supplementary information

   [218]Supplementary Information^ (2.5MB, pdf)
   [219]Peer Review File^ (2.2MB, pdf)
   [220]41467_2024_53611_MOESM3_ESM.pdf^ (383.6KB, pdf)

   Description of Additional Supplementary Files
   [221]Supplementary Data 1^ (18.7KB, xlsx)
   [222]Supplementary Data 2^ (48KB, xlsx)
   [223]Supplementary Data 3^ (22.8KB, xlsx)
   [224]Reporting Summary^ (1.3MB, pdf)

Source data

   [225]Source Data^ (68.7KB, xlsx)

Acknowledgements