**1. Introduction**

Leaves are the main photosynthetic organ of plants. Leaf morphology affects the effective photosynthetic area, which affects accumulation of photosynthetic products and subsequent crop yield. In rice, numerous genes associated with leaf morphogenesis have been mined and cloned, such as *SHALLOT-LIKE 1* (*SLL1*) [1], *HOMEODOMAIN CONTAIN-ING PROTEIN4 (OsHB4)* [2], *SEMI-ROLLED LEAF1*(*SRL1*) [3], *Rice outermost cell-specific gene 5 (Roc5)* [4], *AGO1 homologs 1b* (*OsAGO1b*) [5], *Rice outermost cell-specific 8* (*Roc8*) [6], *PHOTO-SENSITIVE LEAF ROLLING 1* (*PSL1*) [7]. These genes regulate leaf morphogenesis through complex interactions among plant hormone signaling pathways, transcription factors, and microRNAs [8,9]. In addition, leaf morphology is also affected by genes associated with ribosomes synthesis, DNA repair, cell cycle process, cuticle development, ion homeostasis, and microtubule arrangement [9]. However, these genes alone are not sufficient

**Citation:** Wang, J.; Liu, Y.; Hu, S.; Xu, J.; Nian, J.; Cao, X.; Chen, M.; Cen, J.; Liu, X.; Zhang, Z.; et al. *LEAF TIP RUMPLED 1* Regulates Leaf Morphology and Salt Tolerance in Rice. *Int. J. Mol. Sci.* **2022**, *23*, 8818. https://doi.org/10.3390/ ijms23158818

Academic Editor: Ricardo Aroca

Received: 11 July 2022 Accepted: 6 August 2022 Published: 8 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to accurately outline the genetic regulatory network of rice leaf morphogenesis in detail. One of the main challenges in modern agriculture is to increase crop yields under different environmental conditions by cultivating ideal plant architecture [10]. Leaf morphology is an important component of plant architecture and improving it contributes to collaborative improvement of stress resistance and yield. In recent years, great progress has been made in the regulation mechanism of leaf morphology and stress resistance. In addition to the key regulatory roles in plant architecture and yield, many genes regulating leaf morphology also affect characteristics such as drought tolerance, nutrient utilization, and disease resistance. For example, *Ideal Plant Architecture 1* (*IPA1*) not only increases rice yield but also improves rice blast resistance, which counters the traditional view that a single gene cannot simultaneously increase yield and disease resistance [11–14]. *Dwarf 1* (*D1*) is involved in complex network affecting plant height, leaf size, and abiotic stress response [15–17]. *PSL1* regulates rice leaf cell wall development and drought tolerance [7]. Higher leaf temperature, respiration rate, lower transpiration rate, and stomatal conductance in *high temperature susceptibility* (*hts*) resulted in high temperature sensitivity of *hts* [18]. Thus, leaf morphology is closely associated with stress resistance, nutrient utilization, disease resistance, and yield.

Soil salinization is an increasingly serious agricultural problem worldwide [19,20], limiting plant growth and crop productivity in saline–alkali areas [20]. Poor irrigation practices, the improper application of fertilizers, and industrial pollution increased soil salinity in cultivated soil, resulting in aggravated soil salinization [21,22]. Most plants had to develop suitable mechanisms to adjust their physiological and biochemical processes to adapt to high salinity environments during their long evolutionary history due to their sessile nature [20]. Significant progresses have been made for salt tolerance mechanism in plants. They developed suitable strategies to regulate ion and osmotic homeostasis and minimize stress damage [23,24], including exclusion of Na<sup>+</sup> from leaf tissues, compartmentalization of Na<sup>+</sup> (mainly into vacuoles), and reducing water loss while maximizing water absorption [21,25,26]. However, few favorable genetic loci associated with salt resistance have been identified in the breeding practices of rice. Therefore, breeding potentially yieldpenalty-free rice varieties with high salt tolerance is of great significance and an effective way to expand the adaptability and planting area of rice and improve the yield potential of rice in saline–alkali areas.

Wax is the outermost barrier that plays an important role in plant–environment interactions, including plant adaptation to drought environments and various abiotic and biotic stresses. It promotes resistance to ultraviolet (UV) radiation [27] and pests and diseases [28] and protects internal plant tissues from temperature stress [29]. Moreover, the epicuticle wax layer provides the necessary barrier for reducing non-stomatal water loss during drought stress; thereby significantly improve drought tolerance in rice [30,31]. For example, the wax synthesis regulator DROUGHT HYPERSENSITIVE (DHS) interacts with rice outermost cell-specific gene 4 (Roc4), regulating expression of *BODYGUARD* (*BDG*) and thus affecting rice drought tolerance [32,33]. The rice ethylene response factor *WAX SYNTHESIS REGULATORY GENE 1* (*OsWR1*) positively regulates rice wax synthesis and affects drought tolerance by regulating cuticle development and leaf water retention [34]. In addition, wax has a critical effect on the differentiation of plant tissues and organs, such as the morphological development of leaves, fruits, and pollen, thereby affecting plant fertility. Loss function of wax synthesis genes led to morphological abnormalities of flowers and leaves, such as *knb1* (*knobhead*), *bcf1* (*bicentifolia*), and *wax1* in *Arabidopsis* [35]. The *wax2* plants showed disordered leaf structure and fused floral organs in *Arabidopsis* [36]. In rice, most research on wax synthesis genes has focused on pollen development, panicle fertility, and drought resistance; there are few reports on the regulatory role of wax synthesis genes in leaf morphology. Here, we identified a rice mutant *ltr1* with abnormal leaf morphology. This mutant was obtained by ethyl methanesulfonate (EMS) mutagenesis of Nipponbare and was used to isolate and analyze the function of the candidate gene *LTR1* in regulating leaf morphology. We demonstrated that loss function of *LTR1* led to abnormal development of bulliform cells, vascular bundles, and sclerenchyma cells, and to rumpled

leaves, decreases in the seed setting rate and yield, and high sensitivity to salt stress. We also confirmed that *LTR1* mediated regulatory activities of aquaporin and ion transporters result in altered water retention and ion homeostasis under salt stress. Hence, function analysis of *LTR1* in leaf morphology and response to salt stress could provide theoretical foundation for molecular mechanism of leaf morphogenesis and salt response in rice and contribute to breeding efforts to develop salt-tolerant varieties with ideal leaf morphology. mediated regulatory activities of aquaporin and ion transporters result in altered water retention and ion homeostasis under salt stress. Hence, function analysis of *LTR1* in leaf morphology and response to salt stress could provide theoretical foundation for molecular mechanism of leaf morphogenesis and salt response in rice and contribute to breeding efforts to develop salt-tolerant varieties with ideal leaf morphology.

pollen, thereby affecting plant fertility. Loss function of wax synthesis genes led to morphological abnormalities of flowers and leaves, such as *knb1* (*knobhead*), *bcf1* (*bicentifolia*), and *wax1* in *Arabidopsis* [35]. The *wax2* plants showed disordered leaf structure and fused floral organs in *Arabidopsis* [36]. In rice, most research on wax synthesis genes has focused on pollen development, panicle fertility, and drought resistance; there are few reports on the regulatory role of wax synthesis genes in leaf morphology. Here, we identified a rice mutant *ltr1* with abnormal leaf morphology. This mutant was obtained by ethyl methanesulfonate (EMS) mutagenesis of Nipponbare and was used to isolate and analyze the function of the candidate gene *LTR1* in regulating leaf morphology. We demonstrated that loss function of *LTR1* led to abnormal development of bulliform cells, vascular bundles, and sclerenchyma cells, and to rumpled leaves, decreases in the seed setting rate and yield, and high sensitivity to salt stress. We also confirmed that *LTR1*

#### **2. Results 2. Results**

#### *2.1. Identification of the ltr1 Mutant 2.1. Identification of the ltr1 Mutant*

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The *ltr1* mutant was successfully obtained by EMS mutagenesis of the NPB. Phenotypic observation indicated that *ltr1* exhibited abnormal leaf morphology with uneven distribution of bulliform cell on adaxial surface and sclerenchyma cells on abaxial surface and disordered vascular bundles (Figure 1a–c). The contents of chlorophyll *a*, chlorophyll *b*, and carotenoids were significantly higher in *ltr1* than in NPB, with increases of 19.23%, 24.96%, and 17.83%, respectively (Figure 1d). The SPAD (soil and plant analyzer development) value of *ltr1* was significantly higher than that of NPB (Figure 1e). The quantum efficiency of photosystem II (Fv/Fm) and leaf water content of *ltr1* were significantly lower than those of NPB, decreased by 8.69% and 5.26%, respectively (Figure 1f,g). These results showed that growth and development of *ltr1* were seriously impaired. The abnormal leaf morphology of *ltr1* was associated with lower light energy conversion efficiency of the PS II (Photosystem II) reaction center and poor leaf water retention. *The ltr1* mutant was successfully obtained by EMS mutagenesis of the NPB. Phenotypic observation indicated that *ltr1* exhibited abnormal leaf morphology with uneven distribution of bulliform cell on adaxial surface and sclerenchyma cells on abaxial surface and disordered vascular bundles (Figure 1a–c). The contents of chlorophyll *a*, chlorophyll *b*, and carotenoids were significantly higher in *ltr1* than in NPB, with increases of 19.23%, 24.96%, and 17.83%, respectively (Figure 1d). The SPAD (soil and plant analyzer development) value of *ltr1* was significantly higher than that of NPB (Figure 1e). The quantum efficiency of photosystem II (Fv/Fm) and leaf water content of *ltr1* were significantly lower than those of NPB, decreased by 8.69% and 5.26%, respectively (Figure 1f,g). These results showed that growth and development of *ltr1* were seriously impaired. The abnormal leaf morphology of *ltr1* was associated with lower light energy conversion efficiency of the PS II (Photosystem II) reaction center and poor leaf water retention.

**Figure 1.** Phenotype analysis of NPB and *ltr1* plants. (**a**) Plant morphology (bar = 20.0 cm), (**b**) leaf morphology (bar = 4.0 cm), and (**c**) observation of frozen sections of NPB and *ltr1* (bar = 200 µm), red arrows in (**c**) represent bulliform cells. (**d**) Chlorophyll content, (**e**) SPAD, (**f**) Fv/Fm, and (**g**) leaf water content of NPB and *ltr1*. Data are given as means ± SD. Asterisks indicate significant difference based on the Student's *t*-test: \*\* in the figure represents significant difference at *p* < 0.01.

#### *2.2. Effect of LTR1 on Photosynthetic Efficiency and Seed Setting Rate*

According to our results, the panicle length (Figure 2a,b), seed setting rate (Figure 2f), secondary branch numbers (Figure 2e), and grain yield per plant (Figure 2h)were significantly lower in *ltr1* plants than in the wild type, by 14.50%, 95.54%, 16.67%, and 84.49%, respectively. The effective panicle number was significantly higher for *ltr1* than for NPB, with a 43.38% increase (Figure 2c). The primary branch numbers and 1000-grain weight showed no significant differences (Figure 2d,g). These results indicated that the decrease of

ity.

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*2.2. Effect of LTR1 on Photosynthetic Efficiency and Seed Setting Rate*

**Figure 1.** Phenotype analysis of NPB and *ltr1* plants. (**a**) Plant morphology (bar = 20.0 cm), (**b**) leaf morphology (bar = 4.0 cm), and (**c**) observation of frozen sections of NPB and *ltr1* (bar = 200 μm), red arrows in (**c**) represent bulliform cells. (**d**) Chlorophyll content, (**e**) SPAD, (**f**) Fv/Fm, and (**g**) leaf water content of NPB and *ltr1*. Data are given as means ± SD. Asterisks indicate significant difference based on the Student's *t*–test: \*\* in the figure represents significant difference at *p* < 0.01.

According to our results, the panicle length (Figure 2a,b), seed setting rate (Figure 2f), secondary branch numbers (Figure 2e), and grain yield per plant (Figure 2h)were significantly lower in *ltr1* plants than in the wild type, by 14.50%, 95.54%, 16.67%, and 84.49%, respectively. The effective panicle number was significantly higher for *ltr1* than for NPB, with a 43.38% increase (Figure 2c). The primary branch numbers and 1000-grain weight showed no significant differences (Figure 2d,g). These results indicated that the decrease of yield per plant in *ltr1* was caused by the extremely low seed-setting rate and showed that *ltr1* had serious defects in leaf morphology and fertil-

Photosynthesis is the sum of a series of complex metabolic reactions [37]. Maintaining high chlorophyll content in leaves is not necessary to improve the effective photosynthetic rate. Light intensity under low-light conditions is a limiting factor for leaf photosynthesis, and high chlorophyll content is conducive to light absorption; the photosynthetic rate under saturated light intensity is mainly affected by the catalytic ability of the Rubisco enzyme, rather than the limitation of electron transfer rate in light reactions [38]. To explore whether the increased chlorophyll content and abnormal leaf morphology of *ltr1* affect photosynthetic efficiency, we measured the photosynthetic efficiency of NPB and *ltr1* in the field. Compared to NPB, the intercellular CO<sup>2</sup> concentration of *ltr1* was 4.91% higher, and the photosynthetic efficiency was 25.33% lower (Figure 2i,j). Although the photosynthetic pigment content of *ltr1* increased, the photosynthetic efficiency did not. The reasons for the decrease of photo-

yield per plant in *ltr1* was caused by the extremely low seed-setting rate and showed that *ltr1* had serious defects in leaf morphology and fertility. synthetic efficiency in *ltr1* require further exploration.

**Figure 2.** Comparisons of yield characters in NPB and *ltr1*. (**a**) Spike morphology, bar = 4 cm, (**b**) panicle length, (**c**) numbers of effective panicle, (**d**) number of primary branches, (**e**) number of secondary branches, (**f**) seed setting rate, (**g**) 1000-grain weight, (**h**) grain yield per plant, (**i**) photosynthetic efficiency, and (**j**) intercellular CO<sup>2</sup> concentration of NPB and *ltr1*. Data are given as **Figure 2.** Comparisons of yield characters in NPB and *ltr1*. (**a**) Spike morphology, bar = 4 cm, (**b**) panicle length, (**c**) numbers of effective panicle, (**d**) number of primary branches, (**e**) number of secondary branches, (**f**) seed setting rate, (**g**) 1000-grain weight, (**h**) grain yield per plant, (**i**) photosynthetic efficiency, and (**j**) intercellular CO<sup>2</sup> concentration of NPB and *ltr1*. Data are given as means ± SD. Asterisks indicate significant difference based on the Student's *t*-test: \*\* in the figure represents significant difference at *p* < 0.01 and ns in the figure represents there is no significant different at *p* < 0.05.

Photosynthesis is the sum of a series of complex metabolic reactions [37]. Maintaining high chlorophyll content in leaves is not necessary to improve the effective photosynthetic rate. Light intensity under low-light conditions is a limiting factor for leaf photosynthesis, and high chlorophyll content is conducive to light absorption; the photosynthetic rate under saturated light intensity is mainly affected by the catalytic ability of the Rubisco enzyme, rather than the limitation of electron transfer rate in light reactions [38]. To explore whether the increased chlorophyll content and abnormal leaf morphology of *ltr1* affect photosynthetic efficiency, we measured the photosynthetic efficiency of NPB and *ltr1* in the field. Compared to NPB, the intercellular CO<sup>2</sup> concentration of *ltr1* was 4.91% higher, and the photosynthetic efficiency was 25.33% lower (Figure 2i,j). Although the photosynthetic pigment content of *ltr1* increased, the photosynthetic efficiency did not. The reasons for the decrease of photosynthetic efficiency in *ltr1* require further exploration.

#### *2.3. Map-Based Cloning of LTR1*

To explore the molecular mechanism of the phenotype in *ltr1*, an F<sup>2</sup> segregation population was developed by crossing *ltr1* and the *indica* cultivar TN1. The segregation of wild type and mutant phenotype displayed a ratio of 3:1 (Table S1), indicating that the mutant phenotype was controlled by a single recessive gene. Using 21 F<sup>2</sup> mutant individuals, the *LTR1* locus was first mapped to the region between RM6318 and RM1920 on the long arm of chromosome 2. The location was then narrowed down to a 13.5-kb genomic region between the markers N-12 and N-20 (Figure 3a). In this region, only one putative opening reading frame (ORF) was found based on data from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu accessed on 24 March 2021) database.

DNA sequence analysis of the ORF in *ltr1* and NPB revealed that a 2-bp deletion in exon 8 of *LOC\_Os02g40784*, which resulted in a frameshift mutation and early termination of transcription (Figure 3b). *LOC\_Os02g40784* includes ten exons and nine introns and encodes a polypeptide 619 amino acid in length. We therefore inferred that *LOC\_Os02g40784* was the gene controlling the mutant phenotype of *ltr1*. which resulted in a frameshift mutation and early termination of transcription (Figure 3b). *LOC\_Os02g40784* includes ten exons and nine introns and encodes a polypeptide 619 amino acid in length. We therefore inferred that *LOC\_Os02g40784* was the gene controlling the mutant phenotype of *ltr1*.

means ± SD. Asterisks indicate significant difference based on the Student's *t*-test: \*\* in the figure represents significant difference at *p* < 0.01 and ns in the figure represents there is no significant

To explore the molecular mechanism of the phenotype in *ltr1*, an F<sup>2</sup> segregation population was developed by crossing *ltr1* and the *indica* cultivar TN1. The segregation of wild type and mutant phenotype displayed a ratio of 3:1 (Table S1), indicating that the mutant phenotype was controlled by a single recessive gene. Using 21 F<sup>2</sup> mutant individuals, the *LTR1* locus was first mapped to the region between RM6318 and RM1920 on the long arm of chromosome 2. The location was then narrowed down to a 13.5-kb genomic region between the markers N-12 and N-20 (Figure 3a). In this region, only one putative opening reading frame (ORF) was found based on data from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu) database. DNA sequence analysis of the ORF in *ltr1* and NPB revealed that a 2-bp deletion in exon 8 of *LOC\_Os02g40784*,

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 5 of 19

different at *p* < 0.05.

*2.3. Map-Based Cloning of LTR1*

**Figure 3.** Map-based cloning of *LTR1*. (**a**) Fine mapping of *LTR1*; the red arrow represents the mutation site of *LTR1* in *ltr1*. (**b**) Sequence analysis of NPB and *ltr1*; the red box represents the mutation site in *ltr1*. (**c**) Complementary analysis of *LTR1* in *ltr1*; bar for plants and leaves was 20 cm and 5 cm, respectively.

To confirm that the phenotype of *ltr1* was attributable to the detected mutation in *LTR1*, we constructed a complementation vector with a NPB genomic fragment containing the entire coding region of *LTR1* and obtained complementary plants of *LOC\_Os02g40784* under *ltr1* background. As expected, the complementary transgenic T<sup>0</sup> plants showed normal flat leaves: this indicated that the normal expression of *LOC\_Os02g40784* in *ltr1* can complement the phenotype of the mutant (Figure 3c).

#### *2.4. Overexpression and Targeted Deletion of LTR1 2.4. Overexpression and Targeted Deletion of LTR1*

and 5 cm, respectively.

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We next used CRISPR/Cas9 to generate mutant alleles of *LTR1* alleles in a NPB background. We obtained three independent transgenic lines that all carried homozygous mutants, including 3-bp, 4-bp, and 5-bp deletions in exon 3, respectively (Figure 4a,e). These lines had comparable phenotypes to those of *ltr1* with shrunken and distorted leaves, uneven distribution of bulliform cells on adaxial surface and sclerenchyma cells on abaxial surface, and disordered vascular bundles (Figure 4a–d). We also generated overexpression line of *LTR1* in the NPB background, which exhibited longer leaves and higher relative expression level (Figure 5a–c). These results showed that *LOC\_Os02g40784* was *LTR1* and that the mutation in *LOC\_Os02g40784* led to rumpled leaf phenotype in *ltr1*. Moreover, we found that compared with NPB, the grain yield per plant in overexpression of *LTR1* increased by 38.59% (*p* < 0.05), but the grain yields per plant in *ltr1* and *LTR1-KO* decreased by 82.13% and 76.31% (*p* < 0.05), respectively (Figure S6), suggesting that overexpression of *LTR1* enhanced yield in rice. We next used CRISPR/Cas9 to generate mutant alleles of *LTR1* alleles in a NPB background. We obtained three independent transgenic lines that all carried homozygous mutants, including 3-bp, 4-bp, and 5-bp deletions in exon 3, respectively (Figure 4a,e). These lines had comparable phenotypes to those of *ltr1* with shrunken and distorted leaves, uneven distribution of bulliform cells on adaxial surface and sclerenchyma cells on abaxial surface, and disordered vascular bundles (Figure 4a–d). We also generated overexpression line of *LTR1* in the NPB background, which exhibited longer leaves and higher relative expression level (Figure 5a–c). These results showed that *LOC\_Os02g40784* was *LTR1*and that the mutation in *LOC\_Os02g40784* led to rumpled leaf phenotype in *ltr1*. Moreover, we found that compared with NPB, the grain yield per plant in overexpression of *LTR1* increased by 38.59% (*p* < 0.05), but the grain yields per plant in *ltr1* and *LTR1-KO* decreased by 82.13% and 76.31% (*p* < 0.05), respectively (Figure S6), suggesting that overexpression of *LTR1* enhanced yield in rice.

**Figure 3.** Map-based cloning of *LTR1*. (**a**) Fine mapping of *LTR1*; the red arrow represents the mutation site of *LTR1* in *ltr1*. (**b**) Sequence analysis of NPB and *ltr1*; the red box represents the mutation site in *ltr1*. (**c**) Complementary analysis of *LTR1* in *ltr1*; bar for plants and leaves was 20 cm

To confirm that the phenotype of *ltr1* was attributable to the detected mutation in *LTR1*, we constructed a complementation vector with a NPB genomic fragment containing the entire coding region of *LTR1* and obtained complementary plants of *LOC\_Os02g40784* under *ltr1* background. As expected, the complementary transgenic T<sup>0</sup> plants showed normal flat leaves: this indicated that the normal expression of

*LOC\_Os02g40784* in *ltr1* can complement the phenotype of the mutant (Figure 3c).

**Figure 4.** Phenotypic investigation of *LTR1* knockout lines. (**a**) Photos of leaves in NPB, *ltr1*, and *LTR1-KO* lines, bar = 8 cm. (**b**,**c**) Frozen section analysis of leaf in NPB, *ltr1*, and *LTR1-KO* lines; the red arrow represents bulliform cells, and the blue arrow represents the location of sclerenchyma cells. Right of (**b**) is the enlarged detail of red box in the left of (**b**), bar = 200 μm. Right of (**c**) is the enlarged detail of **Figure 4.** Phenotypic investigation of *LTR1* knockout lines. (**a**) Photos of leaves in NPB, *ltr1*, and *LTR1-KO* lines, bar = 8 cm. (**b**,**c**) Frozen section analysis of leaf in NPB, *ltr1*, and *LTR1-KO* lines; the red arrow represents bulliform cells, and the blue arrow represents the location of sclerenchyma cells. Right of (**b**) is the enlarged detail of red box in the left of (**b**), bar = 200 µm. Right of (**c**) is the enlarged detail of red box in the left of (**c**), bar = 100 µm. (**d**) The area of bulliform cells of *LTR1* knockout lines. (**e**) Sequence analysis of WT and *LTR1-KO*. Data are given as means ± SD. Significant differences were determined by Duncan's new multiple range test and indicated with different lowercase letters (*p* < 0.05).

To examine the expression pattern of *LTR1* in NPB, total RNA was extracted from roots, stem, leaf, sheath, and panicles. The qRT-PCR showed that *LTR1* was constitutively expressed in all of the tested tissues, with a dramatic increase in leaves and panicles (Figure 5d). The results were consistent with those of β-glucuronidase (GUS) staining (Figure 5e) and the decreased seed-setting rate of *ltr1* (Figure 2f), showing the important regulatory role of *LTR1* in leaf and panicle development.

**Figure 5.** Overexpression and expression pattern analysis of *LTR1*. (**a**) Phenotypic investigation of overexpression lines of *LTR1*, bar = 6 cm. (**b**) Leaf length of overexpression lines. (**c**) The relative expression level of *LTR1* in overexpression lines. (**d**) The relative expression level of *LTR1* in different organs of NPB. (**e**) Promoter activities of *LTR1* in different organs of NPB as determined by promoter–GUS assays. Data are given as means ± SD. Significant differences were determined by Duncan's new multiple range test and indicated with different lowercase letters ( *p* < 0.05). **Figure 5.** Overexpression and expression pattern analysis of *LTR1*. (**a**) Phenotypic investigation of overexpression lines of *LTR1*, bar = 6 cm. (**b**) Leaf length of overexpression lines. (**c**) The relative expression level of *LTR1* in overexpression lines. (**d**) The relative expression level of *LTR1* in different organs of NPB. (**e**) Promoter activities of *LTR1* in different organs of NPB as determined by promoter– GUS assays. Data are given as means ± SD. Significant differences were determined by Duncan's new multiple range test and indicated with different lowercase letters (*p* < 0.05).

red box in the left of (**c**), bar = 100 μm. (**d**) The area of bulliform cells of *LTR1* knockout lines. (**e**) Sequence analysis of WT and *LTR1-KO*. Data are given as means ± SD. Significant differences were determined by Duncan's new multiple range test and indicated with different lowercase letters ( *p* < 0.05).

#### To examine the expression pattern of *LTR1* in NPB, total RNA was extracted from *2.5. Phylogenetic Analysis of LTR1*

roots, stem, leaf, sheath, and panicles. The qRT-PCR showed that *LTR1* was constitutively expressed in all of the tested tissues, with a dramatic increase in leaves and panicles (Figure 5d). The results were consistent with those of β-glucuronidase (GUS) staining (Figure 5e) and the decreased seed-setting rate of *ltr1* (Figure 2f), showing the important regulatory role of *LTR1* in leaf and panicle development. *2.5. Phylogenetic Analysis of LTR1* Protein domain predictions using NCBI CD Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi accessed on 10 August 2018) showed that LTR1 contained ERG3 (elicitor-responsive genes, ERG) and wax2\_C domains. BLAST-P analysis of the NCBI database showed that LTR1 was highly conserved in higher plants including *Oryza brachyantha* (92.25%), *Brachypodium distachyon* (84.98%), *Aegilops tauschii* (84.87%), *Triticum aestivum* (83.84%), *Setaria italic* (82.90%*), Panicum hallii* Protein domain predictions using NCBI CD Search (https://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi accessed on 10 August 2018) showed that LTR1 contained ERG3 (elicitor-responsive genes, ERG) and wax2\_C domains. BLAST-P analysis of the NCBI database showed that LTR1 was highly conserved in higher plants including *Oryza brachyantha* (92.25%), *Brachypodium distachyon* (84.98%), *Aegilops tauschii* (84.87%), *Triticum aestivum* (83.84%), *Setaria italic* (82.90%*), Panicum hallii* (81.42%*), Sorghum bicolor* (82.23%), and *Zea mays* (78.33%) (Figure S1). To investigate the evolutionary relationships between LTR1 homologs, a phylogenic analysis was performed using the Text Neighbor-Joining Tree method [39]. The results showed that LTR1 is closely related to homologues in the grass family containing *Aegilops tauschii*, *Brachypodium distachyon*, and *Triticum aestivum* (Figure 6 and Figure S1). Overall, these analyses demonstrated that the LTR1 was highly conserved in plants.

#### (81.42%*), Sorghum bicolor* (82.23%), and *Zea mays* (78.33%) (Figure S1). To investigate the *2.6. LTR1 Participates in Water Transport and Ion Homeostasis*

evolutionary relationships between LTR1 homologs, a phylogenic analysis was performed using the Text Neighbor-Joining Tree method [39]. The results showed that LTR1 is closely related to homologues in the grass family containing *Aegilops tauschii*, *Brachypodium distachyon*, and *Triticum aestivum* (Figure 6 and Figure S1). Overall, these analyses demonstrated that the LTR1 was highly conserved in plants. RNA-seq analysis showed that there were 6513 differentially expressed genes (DEGs) in NPB and *ltr1*, of which 3022 were up-regulated and 3480 were down-regulated (Figure S2a and Table S6). There were 118 DEGs related to leaf development, comprising 36 upregulated and 82 down-regulated genes (Figure S3a and Table S7). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were mainly enriched in plant hormone signal transduction pathways, which indicated that *LTR1* may regulate leaf development by participating in hormone signal transduction pathways (Figure S3b). For example, BR C-6 oxidase gene (*OsBR6ox*), *AUXIN RESPONSE FACTOR8* (*OsARF8*), *AUXIN RESPONSE FACTOR17 (OsARF17*), *AUXIN RESPONSE FACTOR16 (OsARF16*), *PHYTOSULFOKINE RECEPTOR 2* (*OsPSKR2*), and *PHYTOSULFOKINE RE-CEPTOR 3 (OsPSKR3*) were up-regulated (Figure S3c) and *PENTATRICOPEPTIDE REPEAT PROTEIN* (*OsPPR6*), *RNA-dependent RNA polymerase 6* (*OsRDR6*), *RNA-directed RNA poly-* *merase 1* (*OsRDR1*), *INCREASED LEAF ANGLE1* (*ILA1*), *dwarf 11* (*d11*), and *GIBBERELLIN 20-OXIDASE GENE* (*OsGA20ox1*) were down-regulated in *ltr1* plants (Figure S3d).

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 8 of 19

abiotic stresses; in particular, Na<sup>+</sup>

**Figure 6.** Phylogenic tree of LTR1 and its homologs. The tree was constructed using MEGA 7.0. Protein sequences are *Oryza sativa Japonica* Group (XP 015627618.1), *Oryza sativa Indica* Group (EEC 73617.1), *Oryza brachyantha* (XP 006647531.1), *Brachypodium distachyon* (XP 003575378.1), *Aegilops tauschii* (XP 020161878.1), *Triticum aestivum* (ACA 14353.1), *Setaria italic* (XP 004953128.1), *Panicum halli*i (XP 025794388.1), *Sorghum bicolor* (XP 002454185.1), *Zea mays* (AQK 72680.1), *Hordeum vulgare*  (ABF 51011.1), *Nicotiana tabacum* (XP 016454385.1), *Arabidopsis thaliana* (NP 001320547.1). Scale represents percentage substitutions per site. Statistical support for the nodes is indicated. **Figure 6.** Phylogenic tree of LTR1 and its homologs. The tree was constructed using MEGA 7.0. Protein sequences are *Oryza sativa Japonica* Group (XP 015627618.1), *Oryza sativa Indica* Group (EEC 73617.1),*Oryza brachyantha* (XP 006647531.1), *Brachypodium distachyon* (XP 003575378.1), *Aegilops tauschii* (XP 020161878.1), *Triticum aestivum* (ACA 14353.1), *Setaria italic* (XP 004953128.1), *Panicum hallii* (XP 025794388.1), *Sorghum bicolor* (XP 002454185.1), *Zea mays* (AQK 72680.1), *Hordeum vulgare* (ABF 51011.1), *Nicotiana tabacum* (XP 016454385.1), *Arabidopsis thaliana* (NP 001320547.1). Scale represents percentage substitutions per site. Statistical support for the nodes is indicated.

*2.6. LTR1 Participates in Water Transport and Ion Homeostasis* RNA-seq analysis showed that there were 6513 differentially expressed genes (DEGs) in NPB and *ltr1*, of which 3022 were up-regulated and 3480 were down-regulated (Figure S2a and Table S6). There were 118 DEGs related to leaf development, comprising 36 up-regulated and 82 down-regulated genes (Figure S3a and Table S7). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were mainly enriched in plant hormone signal transduction pathways, which indicated that *LTR1* may regulate leaf development by participating in hormone signal transduction pathways (Figure S3b). For example, BR C-6 oxidase gene (*OsBR6ox*), *AUXIN RESPONSE FACTOR8* (*OsARF8*), *AUXIN RESPONSE FACTOR17 (OsARF17*), *AUXIN RESPONSE FACTOR16 (OsARF16*), *PHYTOSULFOKINE RECEP-TOR 2* (*OsPSKR2*), and *PHYTOSULFOKINE RECEPTOR 3 (OsPSKR3*) were up-regulated (Figure S3c) and *PENTATRICOPEPTIDE REPEAT PROTEIN* (*OsPPR6*), *RNA-dependent RNA polymerase 6* (*OsRDR6*), *RNA-directed RNA polymerase 1* (*OsRDR1*), *INCREASED LEAF ANGLE1* (*ILA1*), *dwarf 11* (*d11*), and *GIBBERELLIN 20-OXIDASE GENE*  (*OsGA20ox1*) were down-regulated in *ltr1* plants (Figure S3d). A Gene Ontology (GO) term enrichment analysis was also conducted for DEGs A Gene Ontology (GO) term enrichment analysis was also conducted for DEGs between NPB and *ltr1*. The most highly enriched GO biological processes were in salt-stress response, stimulus response, and ABA response (Figure S2b). The most highly enriched GO molecular functions were ATP binding and protein binding, and the most enriched cell components were plasma membrane and nucleus (Figure S2c,d). These results suggested that *LTR1* was involved in the salt-stress response. It was previously reported that plant membrane transporters play key roles in resistance to biological and abiotic stresses; in particular, Na+/K<sup>+</sup> transporters increase resistance to salt stress [40]. We further found that there were 259 up-regulated and 178 down-regulated DEGs related to the salt-stress response (Figure 7a and Table S8). In *ltr1*, most of the genes encoding aquaporin or related to Na+/K<sup>+</sup> transporters were up-regulated, such as *PLASMA MEMBRANE INTRINSIC PROTEIN genes OsPIP1;1*, *OsPIP1;2*, *OsPIP1;3*, *OsPIP2;1*, *OsPIP2;2*, *OsPIP2;4*, *OsPIP2;4*; *TONOPLAST INTRINSIC PROTEIN* genes *OsTIP1;1*, *OsTIP1;2*; *HIGH-AFFINITY K<sup>+</sup> TRANS-PORTERS* genes *OsHKT1;14*, *OsHKT2;3*, and *OsHKT1;5* (Figure 7b). These results suggested that *LTR1* may affect salt tolerance by regulating water transport and ion homeostasis in plants through aquaporin and Na+/K<sup>+</sup> transporters. Given that many genes encoding aquaporins and ion transporter were differentially expressed in NPB and *ltr1*, we considered the possibility that *LTR1* may regulate salt tolerance by affecting water transport and ion homeostasis. Therefore, we measured the Na<sup>+</sup> content in solution and in tissues of NPB

transporters increase resistance to salt stress [40].

results suggested that *LTR1* was involved in the salt-stress response. It was previously reported that plant membrane transporters play key roles in resistance to biological and

/K<sup>+</sup>

between NPB and *ltr1*. The most highly enriched GO biological processes were in

and *ltr1* under salt stress. After salt stress, Na<sup>+</sup> content in stems and leaves of *ltr1* were significantly higher than those of NPB, which increased by 28.24% and 45.75%, respectively (*p* < 0.05). There was no significant difference in Na<sup>+</sup> content in the roots of NPB and *ltr1* (*p*< 0.05) (Figure 7c). Furthermore, there was no significant difference in Na<sup>+</sup> content in the liquid media in which NPB and *ltr1* plants were grown after treatment in hydroponic solution for 1 d (*p* < 0.05). However, after treatment for 3 or 6 d, Na<sup>+</sup> content was lower in the solution in which *ltr1* plants were grown compared to NPB plants, decreased by 15.20% and 8.03%, respectively (*p* < 0.01) (Figure 7d). Under normal growth conditions (CK), the relative expression levels of *OsPIP1;1*, *OsPIP1;2*, *OsPIP2;1*, *OsPIP2;2*, *OsTIP1;1, OsTIP1;2*, *OsHKT1;1*, *OsHKT1;5*, and *OsHKT2;3* were significantly higher in *ltr1* than NPB leaves (Figure 7e), increased by 1.85, 2.36, 3.48, 3.46, 10.2, 2.57, 2.67, 2.52, 3.44 times, respectively (*p* < 0.01); which was consistent with the RNA-seq results. The genes encoding aquaporin and ion transporter in NPB and *ltr1* plants both were strongly induced by salt stress. However, the induction of these genes was stronger in *ltr1* than in leaves of NPB, leading to relative expression levels of *OsPIP1;1*, *OsPIP1;2*, *OsPIP2;1*, *OsPIP2;2*, *OsTIP1;1*, *OsTIP1;2*, *OsHKT1;5*, and *OsHKT2;3* that were significantly higher in *ltr1* than NPB leaves under salt stress (*p* < 0.01), especially the expression of *OsPIP2;1* and *OsHKT2;3* (Figure 7f). This was consistent with the finding that the Na<sup>+</sup> content in stems and leaves of *ltr1* were significantly higher than those of NPB.
