OsLEA1b Modulates Starch Biosynthesis at High Temperatures in Rice

High temperatures accelerate the accumulation of storage material in seeds, often leading to defects in grain filling. However, the mechanisms regulating grain filling at high temperatures remain unknown. Here, we want to explore the quality factors influenced by the environment and have identified a LATE EMBROYGENESIS ABUNDANT gene, OsLEA1b, a heat-stress-responsive gene in rice grain filling. OsLEA1b is highly expressed in the endosperm, and its coding protein localizes to the nucleus and cytoplasm. Knock-out mutants of OsLEA1b had abnormal compound starch granules in endosperm cells and chalky endosperm with significantly decreased grain weight and grain number per panicle. The oslea1b mutants exhibited a lower proportion of short starch chains with degrees of polymerization values from 6 to 13 and a higher proportion of chains with degrees from 14 to 48, as well as significantly lower contents of starch, protein, and lipid compared to the wild type. The difference was exacerbated under high temperature conditions. Moreover, OsLEA1b was induced by drought stress. The survival rate of oslea1b mutants decreased significantly under drought stress treatment, with significant increase in ROS levels. These results indicate that OsLEA1b regulates starch biosynthesis and influences rice grain quality, especially under high temperatures. This provides a valuable resource for genetic improvement in rice grain quality.


Introduction
Rice (Oryza sativa) is an important crop consumed as a staple food by more than half of the world's population.The rice endosperm is the main storage organ for nutrients, including starch, proteins, and lipids [1].Starch is the main endosperm component, and its content, composition, and structure greatly affect grain quality and yield [2]. Sucrose is efficiently converted into starch through a series of reactions during rice endosperm development [3].There are three main types of enzymes involved in starch biosynthesis.Starch synthase (SS) lengthens the non-reducing end of glucose chains; branching enzymes (BEs) then generate branches from the existing chain; and debranching enzymes (DBEs) hydrolyze branches [4].In addition, several non-enzymatic proteins participate in starch biosynthesis in the cereal endosperm, such as PROTEIN TARGETING TO STARCH (PTST) and the starch-binding protein FLOURY ENDOSPERM 6 (FLO6) [5,6].
The environment also affects endosperm starch biosynthesis.Heat-induced crop damage is becoming increasingly common as a result of global warming, severely limiting high and stable rice yields [7].Rice can tolerate certain temperature ranges during Plants 2023, 12, 4070 2 of 15 different developmental stages [8].High temperatures perturb the expression of starch biosynthesis-related genes, hindering starch biosynthesis and adversely affecting rice during the grain-filling period [9].Suppression of OsMADS7, encoding a transcription factor, in rice endosperm stabilizes amylose content under high-temperature stress [10].Inhibiting the expression of α-amylase genes Amy1A, Amy1C, Amy3A, Amy3D, and Amy3E reduces starch degradation and results in chalky grains under high-temperature conditions [11].The high pre-mRNA splicing efficiency of Waxy (Wx) likely stabilizes amylose content in rice seeds under high temperatures [12].Moreover, genes specifically expressed in the endosperm play an important role in crop growth and development.
There are several endosperm-specific genes that were identified through a genomic survey [13].These genes encode grain storage proteins and proteins related to starch biosynthesis, such as the drought-stress-responsive LATE EMBROYGENESIS ABUNDANT (LEA) proteins [14].In Arabidopsis (Arabidopsis thaliana), LEA proteins are associated with seed dehydration tolerance [15].Four NAC transcription factors bind the OsLEA promoter to activate its expression to high levels in embryonic tissues during endosperm development [16].However, the effects of LEA proteins on starch biosynthesis under high-temperature conditions remain unknown.
Here, we want to explore the quality factors influenced by environment and have identified an embryonic protein homologous to OsLEA3-2 [17], which we named OsLEA1b.The oslea1b mutants were more sensitive to high-temperature stress and drought stress than the wild type.Milled rice from oslea1b mutants showed significantly lower starch content and abnormal structure under a high-temperature environment, which resulted in significantly greater chalkiness and chalky grain rate than that from the wild type.A series of assays were utilized to determine the effects of OsLEA1b on starch biosynthesis under high-temperature conditions.These findings enhance our understanding of OsLEA proteins and grain filling in rice, providing useful information for genetic improvement of rice grain yield and quality.

OsLEA1b Encodes an Embryonic Protein Mainly Expressed in the Endosperm
To isolate genes that regulate rice grain filling under high-temperature conditions, we searched the expression of genes that are induced under high temperatures in our RNA-Seq data (Supplementary Figure S1).We identified OsLEA1b (LOC_Os01g16920), which is predominantly expressed during the grain-filling stage and is homologous to OsLEA3-2 (Supplementary Figure S1).Reverse transcription quantitative PCR (RT-qPCR) analysis validated that OsLEA1b is highly expressed in developing seeds, particularly during the late stages of seed development (Figure 1A).In contrast, OsLEA1b was rarely expressed in leaf and stem tissues.We transformed a vector harboring the β-glucuronidase (GUS) reporter gene driven by the OsLEA1b promoter into rice.Histochemical analysis corroborated that GUS activity in transgenic plants was strongest in the endosperm (Figure 1B), consistent with the RT-qPCR results.Transient expression analysis of a 35S:OsLEA1b-GFP construct in rice protoplasts revealed that the OsLEA1b-GFP fusion protein was widely present in the cytoplasm and nucleus, similar to the GFP control (Figure 1C).We therefore concluded that OsLEA1b encodes an embryonic protein.

The oslea1b Mutants Exhibit Chalky Endosperm and Decreased Yield
The OsLEA1b gene contains two exons and one intron and does not harbor any known domains aside from four low-complexity regions (determined using SMART, http://smart.embl-heidelberg.de,4accessed on January 2018, Figure S2).To investigate the function of OsLEA1b in rice endosperm development and plant growth, we knocked out OsLEA1b in the japonica rice cultivar 'ZH11′ using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated genome editing.We successfully obtained two independent homozygous mutants, oslea1b-1 and oslea1b-2.RT-qPCR analysis showed that OsLEA1b expression in the two mutants was significantly lower than that in the wild type (Figure S3A-C).Compared to the wild type, the grain width of oslea1b mutants was significantly lower, and this resulted in a lower 1000-grain weight even though the grains were longer in length (Figure 2A-C,H-K).Moreover, we observed that most mutant grains showed a semi-opaque endosperm, and the grain chalkiness rate in the mutants reached 95% (Figure 2D-G,O).A dramatically lower grain number per panicle in the mutants resulted in lower yields, only 49% relative to the wild-type control.But panicle length, tiller number, and seed setting rate were not significantly different in the mutants compared to the wild type (Figure 2L-N, Figure S4).Besides the shorter plant height, the grain-filling rate in the oslea1b mutants

The oslea1b Mutants Exhibit Chalky Endosperm and Decreased Yield
The OsLEA1b gene contains two exons and one intron and does not harbor any known domains aside from four low-complexity regions (determined using SMART, http://smart.embl-heidelberg.de,accessed on 1 January 2018, Figure S2).To investigate the function of OsLEA1b in rice endosperm development and plant growth, we knocked out OsLEA1b in the japonica rice cultivar 'ZH11 using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated genome editing.We successfully obtained two independent homozygous mutants, oslea1b-1 and oslea1b-2.RT-qPCR analysis showed that OsLEA1b expression in the two mutants was significantly lower than that in the wild type (Figure S3A-C).Compared to the wild type, the grain width of oslea1b mutants was significantly lower, and this resulted in a lower 1000-grain weight even though the grains were longer in length (Figure 2A-C,H-K).Moreover, we observed that most mutant grains showed a semi-opaque endosperm, and the grain chalkiness rate in the mutants reached 95% (Figure 2D-G,O).A dramatically lower grain number per panicle in the mutants resulted in lower yields, only 49% relative to the wild-type control.But panicle length, tiller number, and seed setting rate were not significantly different in the mutants compared to the wild type (Figures 2L-N and S4).Besides the shorter plant height, the grain-filling rate in the oslea1b mutants was slower on the 20th and 30th days after fertilization (DAF) than that of the wild type (Figures S4 and S5).Thus, the OsLEA1b gene plays an important role in grain quality and yield.
Plants 2023, 12, x FOR PEER REVIEW 4 of 15 was slower on the 20th and 30th days after fertilization (DAF) than that of the wild type (Figures S4 and S5).Thus, the OsLEA1b gene plays an important role in grain quality and yield.

OsLEA1b Affects Starch Granule Development in the Endosperm
We next observed the starch grain morphology in endosperm cells of oslea1b mutants and the wild type.Scanning electron microscopy (SEM) revealed that wild-type starch granules were polygonal in shape and densely arranged.In contrast, starch granules in the chalky endosperm of the mutants were round and had numerous air spaces (Figure Data are means ± SD from at least three biological replicates.Statistically significant differences were determined using Student's t-test (*, p < 0.05, **, p < 0.01).

OsLEA1b Affects Starch Granule Development in the Endosperm
We next observed the starch grain morphology in endosperm cells of oslea1b mutants and the wild type.Scanning electron microscopy (SEM) revealed that wild-type starch granules were polygonal in shape and densely arranged.In contrast, starch granules in the chalky endosperm of the mutants were round and had numerous air spaces (Figure 3A-C).We prepared semi-thin sections of grains on the 10th DAF to observe the morphological differences in complex starch granules in the endosperm.Mutant grains had greater numbers of loosely arranged complex starch granules in peripheral and central endosperm cells than exhibited in wild-type grains (Figure 3D-G).In addition, gaps between amyloplasts in the center of the mutant endosperm were larger than those in wild-type endosperm, with many dispersed starch granules in the mutant (Figure 3H,I).Meanwhile, transmission electron microscopy (TEM) revealed many abnormal starch granules developed in the mutant endosperm (Figure 3J-O).
Plants 2023, 12, x FOR PEER REVIEW 5 of 15 3A-C).We prepared semi-thin sections of grains on the 10th DAF to observe the morphological differences in complex starch granules in the endosperm.Mutant grains had greater numbers of loosely arranged complex starch granules in peripheral and central endosperm cells than exhibited in wild-type grains (Figure 3D-G).In addition, gaps between amyloplasts in the center of the mutant endosperm were larger than those in wildtype endosperm, with many dispersed starch granules in the mutant (Figure 3H,I).Meanwhile, transmission electron microscopy (TEM) revealed many abnormal starch granules developed in the mutant endosperm (Figure 3J-O).We examined additional physicochemical properties of starch from wild-type and oslea1b endosperm.Amylose, total starch, and protein contents in the mutants were significantly lower than those in the wild type (Figure 4A-C).The gel consistency of starch in the wild type was 81 mm, while that of oslea1b mutant starch was 39.5-53 mm (Figure 4D).The distribution of amylopectin chain lengths suggested a lower proportion of short chains with degrees of polymerization values between 6 and 13 but a higher proportion of intermediate chains (degrees 14 to 48) in the oslea1b mutants than that in the wild type (Figure 4E).Starch gelatinization and viscosity analyses indicated that the mutant starch was more difficult to gelatinize (Figure 4F,G).These results show that the physicochemical properties of endosperm starch were altered in the oslea1b mutants.We examined additional physicochemical properties of starch from wild-type and oslea1b endosperm.Amylose, total starch, and protein contents in the mutants were significantly lower than those in the wild type (Figure 4A-C).The gel consistency of starch in the wild type was 81 mm, while that of oslea1b mutant starch was 39.5-53 mm (Figure 4D).The distribution of amylopectin chain lengths suggested a lower proportion of short chains with degrees of polymerization values between 6 and 13 but a higher proportion of intermediate chains (degrees 14 to 48) in the oslea1b mutants than that in the wild type (Figure 4E).Starch gelatinization and viscosity analyses indicated that the mutant starch was more difficult to gelatinize (Figure 4F,G).These results show that the physicochemical properties of endosperm starch were altered in the oslea1b mutants.

The oslea1b Mutants Showed Lower Grain Quality under High Temperature Conditions
High temperature during rice grain filling often reduces grain quality [18].We grew wild-type and oslea1b plants under field conditions until flowering, after which plants were subjected to artificial high temperatures (35 • C for 12 h in light and 28 • C for 12 h in darkness) and normal temperatures (28 • C for 12 h in light and 22 • C for 12 h in darkness).Under high-temperature conditions, mature wild-type grains were slightly chalky, but mature oslea1b grains were floury with a significantly lower 1000-grain weight (Figure 5A-H).Compared to those under normal temperature conditions, wild-type and oslea1b grains had significantly lower amylose and total protein contents under hightemperature conditions.However, the differences between conditions were greater for oslea1b grains than for wild-type grains (Figure 5I-K).

The oslea1b Mutants Showed Lower Grain Quality under High Temperature Conditions
High temperature during rice grain filling often reduces grain quality [18].We grew wild-type and oslea1b plants under field conditions until flowering, after which plants were subjected to artificial high temperatures (35 °C for 12 h in light and 28 °C for 12 h in darkness) and normal temperatures (28 °C for 12 h in light and 22 °C for 12 h in darkness).Under high-temperature conditions, mature wild-type grains were slightly chalky, but mature oslea1b grains were floury with a significantly lower 1000-grain weight (Figure 5A-H).Compared to those under normal temperature conditions, wild-type and oslea1b grains had significantly lower amylose and total protein contents under high-temperature conditions.However, the differences between conditions were greater for oslea1b grains than for wild-type grains (Figure 5I-K).We analyzed the expression of genes related to starch biosynthesis under high and normal temperatures.High temperature induced OsLEA1b expression at the early grainfilling stage (Figure S6).The expression levels of STARCH BRANCHING ENZYME 1 (OsBE1) and BRITTLE 1 (OsBT1) were significantly lower in the mutant than in the wild type under high temperature conditions.Conversely, the expression levels of SOLU-BLE STARCH SYNTHASE IIA (OsSSIIa), PULLULANASE (PUL), ISOAMYLASE 3 (ISA3), STARCH BRANCHING ENZYME IIb (OsBEIIb), GRANULE-BOUND STARCH SYNTHASE I (GBSS1), RAG2, and GLUTELIN TYPE-D 1 (GLUD1) were significantly higher in the mutant than in the wild type under high temperatures (Figure 6).These differences in gene expression likely led to the differences observed in amylopectin's fine structure in the mutants under high-temperature conditions.
Plants 2023, 12, x FOR PEER REVIEW 8 of 15 We analyzed the expression of genes related to starch biosynthesis under high and normal temperatures.High temperature induced OsLEA1b expression at the early grainfilling stage (Figure S6).The expression levels of STARCH BRANCHING ENZYME 1 (OsBE1) and BRITTLE 1 (OsBT1) were significantly lower in the mutant than in the wild type under high temperature conditions.Conversely, the expression levels of SOLUBLE STARCH SYNTHASE IIA (OsSSIIa), PULLULANASE (PUL), ISOAMYLASE 3 (ISA3), STARCH BRANCHING ENZYME IIb (OsBEIIb), GRANULE-BOUND STARCH SYNTHASE I (GBSS1), RAG2, and GLUTELIN TYPE-D 1 (GLUD1) were significantly higher in the mutant than in the wild type under high temperatures (Figure 6).These differences in gene expression likely led to the differences observed in amylopectin's fine structure in the mutants under high-temperature conditions.

OsLEA1b Promotes Drought Tolerance
The oslea1b mutants were sensitive to high temperature.We also examined the sensitivity of the oslea1b mutants to abiotic stresses such as drought and salinity.Treatment with 20% PEG6000 or 200 mM NaCl for 1 week induced greater reductions of root and shoot length in the mutant than in the wild type.Moreover, the survival rates of oslea1b Plants 2023, 12, 4070 9 of 15 mutants were only 13% and 11% when subjected to 20% PEG6000 (Figures 7A-C and S7), indicating that the mutants were more sensitive to osmotic stress.When we exposed two-week-old seedlings to drought stress under soil conditions, the survival rate of oslea1b mutants was obviously reduced to 10% compared to that of the wild type (Figure 7D,E).OsLEA1b expression was induced after 12 h of 20% PEG6000 treatment in wild-type plants but not in the mutants (Figure 7F).These results indicate that the oslea1b mutants have reduced tolerance to drought stress.

OsLEA1b Promotes Drought Tolerance
The oslea1b mutants were sensitive to high temperature.We also examined the sensitivity of the oslea1b mutants to abiotic stresses such as drought and salinity.Treatment with 20% PEG6000 or 200 mM NaCl for 1 week induced greater reductions of root and shoot length in the mutant than in the wild type.Moreover, the survival rates of oslea1b mutants were only 13% and 11% when subjected to 20% PEG6000 (Figure 7A-C, Figure S7), indicating that the mutants were more sensitive to osmotic stress.When we exposed two-week-old seedlings to drought stress under soil conditions, the survival rate of oslea1b mutants was obviously reduced to 10% compared to that of the wild type (Figure 7D,E).OsLEA1b expression was induced after 12 h of 20% PEG6000 treatment in wild-type plants but not in the mutants (Figure 7F).These results indicate that the oslea1b mutants have reduced tolerance to drought stress.(D,E) Phenotypes of WT and oslea1b before and after drought stress.Water was withheld for 5 d followed by a 14-day recovery period.(F) Relative expression of OsLEA1b was induced by drought stress.(G-J) Peroxidase (POD) enzyme activity (G), and contents of hydrogen peroxide (H 2 O 2 ; H), proline (I), and malondialdehyde (MDA; (J)) in WT and oslea1b mutant leaves.(K,L) Relative expression of genes related to reactive oxygen species scavenging and ABA signaling in wild type and mutants before (K) and after (L) 20% PEG treatment.Data are means ± SD from at least three biological replicates.Different letters in (C, G-J) indicate significant differences at p < 0.05 by ANOVA and Duncan's test.In (K, L), statistically significant differences were determined using Student's t-test (*, p < 0.05, **, p < 0.01).
Reactive oxygen species (ROS) levels were determined in leaves of the wild type and the oslea1b mutants.There were no differences in various ROS indexes between control and stress treatments in the wild type.But oslea1b mutants had significantly increased peroxidase (POD) activity, hydrogen peroxide (H 2 O 2 ) content, proline content, and malondialdehyde (MDA) content after PEG treatment compared to the wild type (Figure 7G-J).Genes related to stress, ROS scavenging, and ABA signaling were significantly up-regulated in the oslea1b mutants after PEG treatment but not in the control (Figure 7K,L).These results indicate that the oslea1b mutants are more sensitive to drought stress than the wild type.

The oslea1b Mutants Showed Greater Sensitivity to High Temperature
High temperature during the rice grain-filling stage often results in lower quantity and worse quality, such as higher chalkiness and lower grain-setting rate [19].Grain chalkiness affects the appearance of rice.The imbalance of a source-to-sink under high temperature conditions leads to insufficient grain filling in endosperm cells and disordered enzyme activity, which bring a high grain chalkiness rate [20][21][22][23][24].In this study, we observed that grains of oslea1b mutants exhibit high chalkiness rates under normal temperatures and chalky endosperm under high temperatures.The abnormally developed starch granules in the mutant endosperm cells were loosely arranged rather than whole complex granules.The oslea1b mutants were more sensitive to high temperature during endosperm development than wild-type plants.Moreover, the chalkiness rate and chalkiness degree of the mutants were dramatically higher than those of the wild type under both natural and artificial high temperatures.By contrast, the gelatinization and gel consistency of the two mutants were significantly lower than those in the wild type at high temperatures.Recent studies have shown that in addition to starch content, the arrangement of starch granules also affects rice grain cooking and eating qualities.It is likely that the abnormal starch granule development induced by high temperature in oslea1b mutants would affect the cooking and eating qualities.The higher proportion of short starch chain length and easier pasting properties of oslea1b may confer it greater sensitivity to high temperature tolerance.
Starch content and structure directly influence grain appearance such as chalkiness [25].And many genes regulating starch biosynthesis were reported to influence rice chalkiness [26].Chalk5, the major QTL control chalky grain rate of rice, two polymorphic nucleotides on the promoter of Chalk5 affect its expression level.The lower expression level could decrease the chalkiness of rice and increase the percentage of milled rice.Total starch and amylose content in the oslea1b mutants were significantly lower than those in the wild type, especially under high temperatures.Moreover, a significantly different distribution of amylose chain lengths in the mutant led to an altered starch structure.The expression of BE1, SSIIa, PUL, ISA3, and BEIIb showed significant differences between the wild type and mutants under high-temperature treatment, which resulted in differences in the fine structure of starch [27,28].The fine structure of starch affects starch grain expansion and gelatinization, which impacts the cooking performance and taste of rice [29].Gelatinization temperature was higher in the mutants, causing hard, gluey consistency and reduced viscosity.The expression of SSIIa was significantly higher in the mutant than in the wild type under high temperatures, which may be responsible for the reduced viscosity.Although the effects of high temperature on rice quality are well known, additional research is needed to explore the underlying regulatory network to improve rice quality.

OsLEA1b Plays an Important Role in Rice Stress Responses
Phylogenetic analysis showed that the embryonic protein OsLEA1b is homologous to the late embryogenesis abundant protein OsLEA3-2.LEA proteins are widely distributed in rice, Arabidopsis, maize (Zea mays), cotton (Gossypium hirsutum), potato (Solanum tuberosum), and other plant species [30][31][32][33][34].The role of plant LEA proteins in stress tolerance has been well documented.Plants maintain cell membrane stability in low temperature environments through LEA protein biosynthesis [35].In vitro and in vivo experiments demonstrated that soybean (Glycine max) PM2 (LEA3) protects proteins and maintains enzyme activity under unfavorable temperatures [36].In addition, LEA proteins bind to certain ions to maintain osmotic pressure in cells.Two soybean LEA4 proteins, GmPM1 and GmPM9, possess metalbinding properties that reduce abiotic stress-induced oxidative damage [37].
OsLEA1b is highly expressed in seeds yet barely detectable in other rice tissues.However, the drought sensitivity of oslea1b mutants was observed in other parts of the plant, such as the leaves.Additional studies are needed to explain how OsLEA1b, which is enriched in the endosperm, regulates plant stress tolerance.Overall, our research reveals that OsLEA1b regulates starch biosynthesis in the grain endosperm and plays an important role in rice drought tolerance.This study provides useful information for potential genetic improvement of rice yield and grain quality.

Subcellular Localization
The OsLEA1b coding sequence was cloned into the pAN580-GFP vector capable of generating an OsLEA1b-GFP fusion protein.The fusion construct and an empty vector control (PAN580-GFP) were separately transformed into rice protoplasts [53].GFP fluorescence was detected using a confocal laser scanning microscope with 488 nm (Leica TCS SP5, Wetzlar, Germany).

Electron Microscopy
Dry and completely brown rice grains of oslea1b mutants and the wild type were randomly selected.Images were obtained using a HITACHI S3400N scanning electron microscope (Hi-tachi, Tokyo, Japan).Scanning electron microscopy was performed as described previously [54].To examine the development of compound granules, transverse sections (approximately 1-mm thick) of wild-type and oslea1b endosperms at 10 DAF were used to prepare semi-thin sections (800 nm).Samples were stained with I 2 -KI for 5 s and subsequently examined under a light microscope (Nikon Eclipse 80i, Tokyo, Japan; http://www.nikon.comaccessed on 1 January 2018).All treatments were performed as described previously [55].To observe amyloplast ultrastructure, developing seeds (10 DAF) of wild-type and oslea1b plants were analyzed using transmission electron microscopy (H7650; Hitachi).Samples were treated as described previously [56].

Analysis of Endosperm Starch Physicochemical Properties
Total starch and amylose contents were measured using the Megazyme K-TSTA and K-AMYL (Megazyme, Wicklow, Ireland) kits, respectively.Lipid and protein contents in oslea1b mutant and wild type were quantified following a previously established method [54].Gel consistency was measured according to published methods [57].To determine the pasting properties of endosperm starch, 3 g of milled rice flour (0.5 mm or less, 14% moisture) was transferred into a sample vessel containing 25 mL of distilled water.The sample was mixed and assessed using a Rapid Visco Analyzer (RVA Techmaster, Newport Scientific, Narrabeen, Australia).To determine the chain length distributions of amylopectin, 5 mg of rice powder was digested with Pseudomonas amyloderamosa isoamylase (Megazyme) and then analyzed by capillary electrophoresis (PA800 plus pharmaceutical analysis system, Beckman Coulter, Brea, CA, USA).

Data Analysis
Data are presented as the means ± standard deviation (SD), shown by error bars.The chi-square (χ 2 ) test and independent samples t-test (* p < 0.05; ** p < 0.01; NS, not significant) were used for statistical analysis using IBM SPSS Statistics 26 software.

Conclusions
In summary, we identified OsLEA1b, a heat-stress-responsive gene in rice grain filling.OsLEA1b is highly expressed in the endosperm, and its coding protein localizes to the nucleus and cytoplasm.The oslea1b mutants had abnormal compound starch granules in the endosperm cells and chalky endosperm with significantly decreased grain weight and grain number per panicle, as well as significantly lower contents of starch, protein, and lipid compared to the wild type.Moreover, OsLEA1b was induced by high temperature and drought stress.The oslea1b mutants showed more sensitivity to high temperatures.These results indicate that OsLEA1b regulates starch biosynthesis and influences rice grain quality,

Figure 1 .
Figure 1.Expression pattern and subcellular localization of OsLEA1b.(A) Relative OsLEA1b expression levels in various tissues and in developing endosperms at 5, 10, 15, 20, 25, and 30 days after fertilization (DAF).Values are means ± SD from three biological replicates.(B) GUS staining in root, stem, leaf, spikelet, and developing grains expressing ProOsLEA1b:GUS.(C) Subcellular localization of OsLEA1b in rice protoplasts.Confocal microscopy images show OsLEA1b-GFP localized in the nucleus and cytoplasm.GFP signaling, chlorophyll autofluorescence, bright field, and merged images are shown for each construct.Scale bars, 2 mm in (B) and 10 µm in (C).

Figure 1 .
Figure 1.Expression pattern and subcellular localization of OsLEA1b.(A) Relative OsLEA1b expression levels in various tissues and in developing endosperms at 5, 10, 15, 20, 25, and 30 days after fertilization (DAF).Values are means ± SD from three biological replicates.(B) GUS staining in root, stem, leaf, spikelet, and developing grains expressing ProOsLEA1b:GUS.(C) Subcellular localization of OsLEA1b in rice protoplasts.Confocal microscopy images show OsLEA1b-GFP localized in the nucleus and cytoplasm.GFP signaling, chlorophyll autofluorescence, bright field, and merged images are shown for each construct.Scale bars, 2 mm in (B) and 10 µm in (C).

Figure 2 .
Figure 2. Analysis of agronomic traits of oslea1b mutants compared to the wild type.(A-C) Grain length, width, and thickness of wild-type (WT) and oslea1b grains.Scale bars, 6 mm.(D) Appearance of brown rice grains from WT and oslea1b mutant plants.Scale bar, 2 mm.(E-G) Transverse sections of WT (E) and oslea1b (F,G) endosperm.Scale bars, 2 mm.(H-O) Quantification of grain length (H), grain width (I), 1000-grain weight (J), grain thickness (K), seed number per panicle (L), grain yield per plant (M), seed setting rate (N), and chalky grain percentage (O) for WT plants and oslea1b mutants.Data are means ± SD from at least three biological replicates.Statistically significant differences were determined using Student's t-test (*, P< 0.05, **, P< 0.01).

Figure 2 .
Figure 2. Analysis of agronomic traits of oslea1b mutants compared to the wild type.(A-C) Grain length, width, and thickness of wild-type (WT) and oslea1b grains.Scale bars, 6 mm.(D) Appearance of brown rice grains from WT and oslea1b mutant plants.Scale bar, 2 mm.(E-G) Transverse sections of WT (E) and oslea1b (F,G) endosperm.Scale bars, 2 mm.(H-O) Quantification of grain length (H), grain width (I), 1000-grain weight (J), grain thickness (K), seed number per panicle (L), grain yield per plant (M), seed setting rate (N), and chalky grain percentage (O) for WT plants and oslea1b mutants.Data are means ± SD from at least three biological replicates.Statistically significant differences were determined using Student's t-test (*, p < 0.05, **, p < 0.01).

Figure 3 .
Figure 3. Starch granule formation and amyloplast development in endosperm cells of wild-type and oslea1b mutants.(A-C) Scanning electron microscopy of transverse sections of wild-type (A) and oslea1b (B,C) endosperm.(D-I) Semi-thin sections of WT and oslea1b endosperm at 10 days after fertilization (DAF).(D-F) Micrographs showing the peripheral region; (G-I) micrographs showing the central region.Black stars in (D-F) indicate aleurone cells.Arrows in (H,I) indicate broken amyloplasts.(J-L) Transmission electron microscopy shows well-developed amyloplasts in WT endosperm cells (J,M) and broken amyloplasts in oslea1b endosperm cells (K,L,N,O).Scale bars, 15 µm in (A-C), 40 µm in (D-I), 5 µm in (J-L), and 2 µm in (M-O).

Figure 3 .
Figure 3. Starch granule formation and amyloplast development in endosperm cells of wild-type and oslea1b mutants.(A-C) Scanning electron microscopy of transverse sections of wild-type (A) and oslea1b (B,C) endosperm.(D-I) Semi-thin sections of WT and oslea1b endosperm at 10 days after fertilization (DAF).(D-F) Micrographs showing the peripheral region; (G-I) micrographs showing the central region.Black stars in (D-F) indicate aleurone cells.Arrows in (H,I) indicate broken amyloplasts.(J-L) Transmission electron microscopy shows well-developed amyloplasts in WT endosperm cells (J,M) and broken amyloplasts in oslea1b endosperm cells (K,L,N,O).Scale bars, 15 µm in (A-C), 40 µm in (D-I), 5 µm in (J-L), and 2 µm in (M-O).

Figure 4 .
Figure 4. Starch physicochemical properties in the endosperm of oslea1b mutants.(A-D) Total starch content (A), amylose content (B), protein content (C), and gel consistency (D) of wild-type and Osesp1 mutant endosperm.(E) Differences in amylopectin chain length distribution between WT and oslea1b.(F) Pasting properties of WT and oslea1b endosperm starch determined using a Rapid Visco Analyzer (RVA).The gray line indicates temperature changes during the measurements.(G) Gelatinization temperature of endosperm starch.To, Tp, and Tc represent the onset, peak, and concluding gelatinization temperatures, respectively.Data in (A-D, G) are presented as means ± SD from three replicates.Statistically significant differences were determined using Student's t-test (*, P< 0.05, **, P< 0.01).

Figure 4 .
Figure 4. Starch physicochemical properties in the endosperm of oslea1b mutants.(A-D) Total starch content (A), amylose content (B), protein content (C), and gel consistency (D) of wild-type and Osesp1 mutant endosperm.(E) Differences in amylopectin chain length distribution between WT and oslea1b.(F) Pasting properties of WT and oslea1b endosperm starch determined using a Rapid Visco Analyzer (RVA).The gray line indicates temperature changes during the measurements.(G) Gelatinization temperature of endosperm starch.To, Tp, and Tc represent the onset, peak, and concluding gelatinization temperatures, respectively.Data in (A-D,G) are presented as means ± SD from three replicates.Statistically significant differences were determined using Student's t-test (*, p < 0.05, **, p < 0.01).

Figure 5 .
Figure 5.The oslea1b mutants were more sensitive to high temperatures during the grain-filling stage.(A-F) Appearance of wild-type (WT) and oslea1b mutant grains under high-temperature treatment (35 °C for 12 h in light and 28 °C for 12 h in darkness) and normal temperature treatment (28 °C for 12 h in light and 22 °C for 12 h in darkness).Scale bars, 1 cm.(G-K) Chalky grain percentage (G), 1000-grain weight (H), total starch content (I), amylose content (J), and total protein content (K) of WT and oslea1b mutant grains under normal and high temperatures.Different letters indicate significant differences at p < 0.05 by ANOVA and Duncan's test.

Figure 5 .
Figure 5.The oslea1b mutants were more sensitive to high temperatures during the grain-filling stage.(A-F) Appearance of wild-type (WT) and oslea1b mutant grains under high-temperature treatment (35 • C for 12 h in light and 28 • C for 12 h in darkness) and normal temperature treatment (28 • C for 12 h in light and 22 • C for 12 h in darkness).Scale bars, 1 cm.(G-K) Chalky grain percentage (G), 1000-grain weight (H), total starch content (I), amylose content (J), and total protein content (K) of WT and oslea1b mutant grains under normal and high temperatures.Different letters indicate significant differences at p < 0.05 by ANOVA and Duncan's test.

Figure 6 .
Figure 6.Transcription levels of genes related to regulate amylopectin synthesis in endosperm at 10 DAF.(A-I) Relative expression of genes related to amylopectin biosynthesis.Gene expression levels were measured in WT and oslea1b-1 grains at 9 days after fertilization under high (HT) and normal (RT) temperature conditions.Different letters indicate significant differences at p < 0.05 by ANOVA and Duncan's test.

Figure 6 .
Figure 6.Transcription levels of genes related to regulate amylopectin synthesis in endosperm at 10 DAF.(A-I) Relative expression of genes related to amylopectin biosynthesis.Gene expression levels were measured in WT and oslea1b-1 grains at 9 days after fertilization under high (HT) and normal (RT) temperature conditions.Different letters indicate significant differences at p < 0.05 by ANOVA and Duncan's test.

Figure 7 .
Figure 7. OsLEA1b gene positively regulates drought stress tolerance.(A,B) Phenotypes of seedlings of wild-type and oslea1b before and after 20% PEG treatment.(C) Survival rate of seedlings of WT and oslea1b after 20% PEG treatment.(D,E) Phenotypes of WT and oslea1b before and after drought stress.Water was withheld for 5 d followed by a 14-day recovery period.(F) Relative expression of OsLEA1b was induced by drought stress.(G-J) Peroxidase (POD) enzyme activity (G), and contents of hydrogen peroxide (H2O2; H), proline (I), and malondialdehyde (MDA; (J)) in WT and oslea1b mutant leaves.(K,L) Relative expression of genes related to reactive oxygen species scavenging and ABA signaling in wild type and mutants before (K) and after (L) 20% PEG treatment.Data are means ± SD from at least three biological replicates.Different letters in (C, G-J) indicate significant differences at p < 0.05 by ANOVA and Duncan's test.In (K, L), statistically significant differences were determined using Student's t-test (*, P< 0.05, **, P< 0.01).

Figure 7 .
Figure 7. OsLEA1b gene positively regulates drought stress tolerance.(A,B) Phenotypes of seedlings of wild-type and oslea1b before and after 20% PEG treatment.(C) Survival rate of seedlings of WT and oslea1b after 20% PEG treatment.(D,E) Phenotypes of WT and oslea1b before and after drought stress.Water was withheld for 5 d followed by a 14-day recovery period.(F) Relative expression of OsLEA1b was induced by drought stress.(G-J) Peroxidase (POD) enzyme activity (G), and contents of hydrogen peroxide (H 2 O 2 ; H), proline (I), and malondialdehyde (MDA; (J)) in WT and oslea1b mutant leaves.(K,L) Relative expression of genes related to reactive oxygen species scavenging and ABA signaling in wild type and mutants before (K) and after (L) 20% PEG treatment.Data are means ± SD from at least three biological replicates.Different letters in (C, G-J) indicate significant differences at p < 0.05 by ANOVA and Duncan's test.In (K, L), statistically significant differences were determined using Student's t-test (*, p < 0.05, **, p < 0.01).