Penalties in Granule Size Distribution and Viscosity Parameters of Starch Caused by Lodging in Winter Wheat

: Granule size distribution of wheat starch is an important characteristic that could affect the functionality of wheat ( Triticum aestivum L.) products. Lodging is a major limiting factor for wheat production. Few studies have been conducted to clarify how lodging influences the granule size distribution and viscosity parameters of starch in wheat grains. Two growing seasons, two high-yield winter wheat cultivars, and five artificial lodging treatments were imposed. The results indicated that lodging significantly reduced the content of starch and increased that of protein. Additionally, lodging caused a marked drop in both starch and protein yields. The relative loss of grain yield, starch yield, harvest index, and protein yield all differed remarkably among lodging treatments with a ranking of L2 > L1 > L4 > L3. Lodging also led to a reduction in the proportion (both by volume and by surface area) of B-type granules and a corresponding increase in that of A-type granules, and the more serious the lodging degree, the greater effect on the changes in these proportions. The smaller starch granules predominated in number, even though their collective contribution to the overall volume is was relatively minor. Meanwhile, it was found that the peak viscosity, hold viscosity, final viscosity, breakdown viscosity, and rebound value of wheat starch were significantly decreased by lodging. Correlation analysis showed that the peak and final viscosities were negatively correlated with volume percentages of A-type starch granules, but were positively correlated with B-type granules. This indicates that B-type granules have higher peak and final viscosities compared with A-type granules in wheat kernels. Lodging can reduce the proportion of B-type starch granules, and thus reduce the peak and the final viscosity in wheat grain.


Introduction
Wheat (Triticum aestivum L.) stands as one of the most vital crops globally [1].Starch, accounting for 65 to 75% of the grain's final dry weight [2], acts as a versatile component in both food and non-food industries [3][4][5].Environmental elements that influence grain yield and quality, including climatic and agricultural practices, vary from year to year.The quality of wheat flour, affected by genetic and environmental factors, is predominantly dictated by the components and characteristics of starch [6][7][8][9].In wheat grains, starch is found in the form of granules within the endosperm.It is universally accepted that starch is encapsulated within two distinct forms: B-type (<9.9 µm in diameter) and A-type (>9.9 µm) granules [3,[10][11][12].Several reports have indicated that starch granules of varying sizes possess distinct physical, chemical, and functional characteristics [2,4,13,14].The viscosity characteristics of flour are intimately linked to the distribution of starch granule sizes, with large A-type granules exhibiting a lower peak viscosity compared to small ones [6,15,16].
Starch deposition occurs in synchronization with grain development.The formation of A-type granules initiates from the earlier stage of endosperm formation and continues until the end of the endosperm cell division phase, and the B-type granules initiate from approximately 12 days post-anthesis [2,17,18].Hence, there occurs a chronological alteration in the size distribution of starch granules during grain development.Environmental elements, including water availability [7,19], nutrient levels [16], temperature [20,21], light factor [22,23], and soil texture [24], that regulate starch granule size distribution have been extensively studied.For example, the starch content of wheat grains was found to significantly decrease during the grain filling stage due to stress from water scarcity [25].It was also reported that both low light levels post-anthesis [22] and high temperature [2] resulted in decreases in starch content and led to substantial alterations in the size distribution of starch granules within wheat grains.
Lodging, which is the irreversible tilting of a cereal stalk from its typical vertical position, arising from both intrinsic and extrinsic factors, is a common and important problem in primary wheat-producing regions globally [26][27][28].Lodging leads to the plant community being exposed to a multi-stress environment, characterized by damage to the community structure, inadequate ventilation and light penetration, impaired photosynthesis, destruction of the stem's conduction system, and other such issues [29,30].These adverse environmental factors interact with each other, exerting a comprehensive impact on the plants [31,32].Yield losses occur when lodging takes place from the pre-flowering stage to the dough ripening stage; the severity of the yield reduction is mitigated when lodging occurs later, during the grain-filling phase [27, 29,33].Studies involving both natural and artificially-induced lodging have determined that yield losses attributable to lodging can vary from 0 to 80% [29,31,33,34].In wheat fields, stems can partially recover from minor early-stage lodging, but may not recover if severely lodged [31,35].Cultivation practices and experiments have shown that applying more nitrogen fertilizer and increasing planting density are crucial for achieving high yields in wheat [27,32,33].For example, local farmers in the Huang-Huai-Hai Plain of China typically apply a nitrogen rate of around 300 kg ha −1 to attain high productivity in winter wheat production [35].Furthermore, in this local area, the over-application of nitrogen and higher planting density could result in an excessively dense wheat stand, which adversely affects stem growth, and resultantly raises the probability of lodging when confronted with severe weather conditions, such as strong winds and heavy rains [33,36].Notably, the reported yield losses in this area attributed to lodging are between 10% and 57% [27].
From the above, lodging is one of the major constraints limiting high yield of wheat.Nevertheless, there is a scant understanding of how lodging influences the starch composition and starch processing characteristics of wheat grains.Consequently, our current experiments were designed to measure the effects of different degrees of artificial lodging on the granule size distribution and viscosity parameters of starch in wheat grains.All of these artificial approaches ensured the uniformity of lodging conditions for the experimental plants, facilitating the quantification of indicators.The aim of this study was to clarify the effects of transient and permanent stem lodging on the size distribution, composition, and viscosity characteristics of endosperm starch in mature wheat grains.The data gathered will contribute to establishing a theoretical foundation for the targeted application of grains, particularly in regions where wheat production is frequently affected by lodging.

Plant Material and Experimental Design
The field trials were conducted at the Beiluo Experimental Station of Weifang University of Science and Technology, Shouguang, Shandong, China (36 • 85 ′ N, 118 • 79 ′ E) during the 2019-2020 and 2020-2021 growing seasons.This area is characterized by a warm temperate, semi-humid climate with continental monsoon features.Figure 1 illustrates the total monthly rainfall and the average monthly temperature for the two crop-growing seasons.The soil in this area was classified as loam, with a pH level of 6.85.The soil layer ranging from 0 to 200 mm in depth was found to have 76.5 mg kg −1 available nitrogen (N), 23.5 mg kg −1 available phosphate, and 80.5 mg kg −1 available potassium (K).Corn (Zea mays L.) had been the preceding crop, with its straw incorporated back into the field.Furthermore, fertilizers were applied prior to planting, consisting of N (120 kg ha −1 as urea), P (75 kg ha −1 as single superphosphate), and K (120 kg ha −1 as potassium chloride).Additionally, nitrogen was reapplied at the wheat's stem elongation stage (120 kg ha −1 ).

Plant Material and Experimental Design
The field trials were conducted at the Beiluo Experimental Station of Weifang University of Science and Technology, Shouguang, Shandong, China (36°85′ N, 118°79′ E) during the 2019-2020 and 2020-2021 growing seasons.This area is characterized by a warm temperate, semi-humid climate with continental monsoon features.Figure 1 illustrates the total monthly rainfall and the average monthly temperature for the two crop-growing seasons.The soil in this area was classified as loam, with a pH level of 6.85.The soil layer ranging from 0 to 200 mm in depth was found to have 76.5 mg kg −1 available nitrogen (N), 23.5 mg kg −1 available phosphate, and 80.5 mg kg −1 available potassium (K).Corn (Zea mays L.) had been the preceding crop, with its straw incorporated back into the field.Furthermore, fertilizers were applied prior to planting, consisting of N (120 kg ha −1 as urea), P (75 kg ha −1 as single superphosphate), and K (120 kg ha −1 as potassium chloride).Additionally, nitrogen was reapplied at the wheat's stem elongation stage (120 kg ha −1 ).Two winter wheat cultivars currently used in major wheat-producing areas, Jimai22 (JM22) and Jinan17 (JN17), were used.Based on prior research [29][30][31] and our preliminary trials [27,36], we identified that lodging at anthesis has the most severe impact on the wheat growth and yield.Thus, we applied artificial lodging treatments of varying severity to wheat during this critical phase.Five lodging treatments were applied: an un-lodged control (CK); artificial indicated lodging treatments (c.40° and 80° from the vertical position; L1 and L2) applied from anthesis (Digit code [DC] 6.4) to maturity (DC 9.2) [37]; and artificial transient lodging treatments (c.40° and 80° from the vertical position) applied at anthesis (DC 6.4), with the plants being permi ed to regain their vertical orientation naturally after 48 h (L3 and L4).These were imposed by means of bamboo and iron wire that caused the required plant positions (c.40° or 80° from the vertical position, Figure 2).
To keep the control plants upright, bamboo and iron were installed if necessary.Experiments were designed in a split-plot (5 rows, 25 cm apart and 3 m long), with three replications for each cultivar.Seeds were sown on 5 October 2019 and 7 October 2020, using manual planters to achieve a planting density of 1,400,000 plants ha -1 .For each experimental plot, ninety spikes that headed simultaneously were selected and marked.Throughout the experiments, no significant crop harm due to weeds, pests, or diseases was detected.Two winter wheat cultivars currently used in major wheat-producing areas, Jimai22 (JM22) and Jinan17 (JN17), were used.Based on prior research [29][30][31] and our preliminary trials [27,36], we identified that lodging at anthesis has the most severe impact on the wheat growth and yield.Thus, we applied artificial lodging treatments of varying severity to wheat during this critical phase.Five lodging treatments were applied: an un-lodged control (CK); artificial indicated lodging treatments (c.40 • and 80 • from the vertical position; L1 and L2) applied from anthesis (Digit code [DC] 6.4) to maturity (DC 9.2) [37]; and artificial transient lodging treatments (c.40 • and 80 • from the vertical position) applied at anthesis (DC 6.4), with the plants being permitted to regain their vertical orientation naturally after 48 h (L3 and L4).These were imposed by means of bamboo and iron wire that caused the required plant positions (c.40 • or 80 • from the vertical position, Figure 2).

Sampling
Data were collected at grain maturity for every treatment protocol.Two wheat varieties were individually harvested by hand on 9 June 2020 and 13 June 2021, upon reaching maturity.From each plot, fifty marked spikes were selected for analysis, focusing on the content, granule size distribution, and viscosity parameters of starch.The grain yield was measured within a harvest area of 2 m 2 for each plot.

Measurements of Yield-Associated Traits
The determination of starch content involved quantifying the contents of amylose To keep the control plants upright, bamboo and iron were installed if necessary.Experiments were designed in a split-plot (5 rows, 25 cm apart and 3 m long), with three replications for each cultivar.Seeds were sown on 5 October 2019 and 7 October 2020, using manual planters to achieve a planting density of 1,400,000 plants ha -1 .For each experimental plot, ninety spikes that headed simultaneously were selected and marked.Throughout the experiments, no significant crop harm due to weeds, pests, or diseases was detected.

Sampling
Data were collected at grain maturity for every treatment protocol.Two wheat varieties were individually harvested by hand on 9 June 2020 and 13 June 2021, upon reaching maturity.From each plot, fifty marked spikes were selected for analysis, focusing on the content, granule size distribution, and viscosity parameters of starch.The grain yield was measured within a harvest area of 2 m 2 for each plot.

Measurements of Yield-Associated Traits
The determination of starch content involved quantifying the contents of amylose and amylopectin in the wheat grains using a linked spectrophotometric assay as referenced [38], with their combined amounts representing the overall starch content [21].To accomplish this, 100 mg of grain powder was combined with 10 mL of 0.5 mol L −1 KOH solution and heated at 100 • C for 15 min before dilution to 50 mL.An aliquot of 2.5 mL was further diluted, neutralized to pH 3.5 with 0.1 mol L −1 HCl, then treated with 0.5 mL of I 2 -KI solution and made up to a final volume of 50 mL.After 20 min of stirring, absorbance readings were taken at wavelengths of 471, 553, 632, and 740.3 nm using the spectrophotometer.The purified wheat amylose exhibited absorption peaks at 632 and 471 nm, while amylopectin peaks were observed at 740.3 and 553 nm.
To determine the protein content, the procedure of converting grain samples into whole meal wheat flour was carried out using the Senior Mill (Brabender Instruments, South Hackensack, NJ, USA), adhering to the guidelines specified in the AACC 26-50 standard [38].The concentration of nitrogen in the grains (GNC) was measured by employing the semi-micro Kjeldahl method and the AACC method 46-13 [38].The protein content of the grains was then estimated by applying a factor of 5.7 to the GNC [39].
The starch from the wheat varieties was isolated following the procedures outlined by Peng et al. and Li et al. [3,22].First, 2 g of wheat kernels was submerged in 30 mL of double-distilled water at 4 • C for a duration of 24 h.After softening, the seeds underwent degermination, and were then crushed using a traditional mortar and pestle in doubledistilled water, continuing until the majority of the starch granules were liberated.The resulting starch slurry was then passed through a 74 µm mesh screen and subjected to centrifugation at 1700 g for a period of 10 min to precipitate the raw starch.The raw starch underwent a purification process, which was repeated thrice, and each cycle involved the use of 5 mL of 2 mol L −1 NaCl, 0.05 mol L −1 NaOH, 0.07 mol L −1 sodium dodecyl sulphate, and double distilled water, respectively.The starch was rinsed with acetone to eliminate residual water, followed by air-drying at room temperature, and stored at −20 • C.
The particle size distribution of the starch was evaluated using a Coulter Laser LS13320 (Beckman Coulter Instruments, Brea, CA, USA), adhering to the procedures detailed by Li et al. [22].In brief, 50 mg of starch was measured into 10 mL tubes with 5 mL of water, vortexed, and chilled at 4 • C, with vortexing every 20 min.The starch suspension was then analyzed using a laser diffraction particle size analyzer.
To determine the pasting properties of the starch, the RVA-Starch Master2 Analyzer (Perten Instruments, Helsingborg, Sweden) was employed, following the protocols outlined by Li et al. and Minh [16,40].In brief, 3 g of wheat flour was placed into an aluminum container, and 25 mL of distilled water was added and mixed well.The rapid viscosity analyzer was then used to measure the starch viscosity parameters, and each sample was tested three times.

Statistical Analyses
Mean and standard errors were calculated for individual measurements.Analysis of variance was performed with the DPSv7.05Data Processing System (Hangzhou RuiFeng Information Technology Co., Ltd., Hangzhou, China).A least significant differences (LSD) test was employed to assess differences between treatments at the 0.05 probability level.Pearson's correlations were calculated to determine the relationship between the volume percentage and properties of starch.Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA) and SigmaPlot 10.0 (Systat Software Inc., Richmond, CA, USA) software were used for data analyses and post-processing.

Crop Development and Lodging
In the experimental plots, the stems of the L1 and L2 treatments remained in a lodged state from anthesis to maturity, and the stems of the L3 and L4 treatments were bent for 48 h and then were permitted to resume their normal orientation.The phenotypic performance of the plants was similar for both wheat genotypes, and near to 10 • and 30 • (mean of both genotypes) from the vertical, for L3 and L4, respectively.For both genotypes, the lodging degree of plants that were permitted to naturally realign to their vertical posture after 48 h under the L4 treatment was higher than those under the L3 treatment.As anticipated, the plants in the control groups of both varieties remained erect throughout the duration of our research.During the grain filling period, the midday canopy temperatures averaged across the two genotypes for L1, L2, L3, and L4 rose by c. 0.5, 1.2, 0.3, and 0.6 • C by occurrence of lodging, respectively.These increases in canopy temperature among the lodged plants during the post-anthesis phase led to negligible variations in maturity timing, with discrepancies of one day or less.

Grain Quality and Yield
Lodging and year had significant effects on amylose content, starch content, and protein content, but the effect of their interaction was not significant (Table 1).In contrast to the un-lodged controls, lodging significantly reduced the content of amylose (Figure 3A,B) and starch (Figure 3C,D), but increased that of protein (Figure 3E,F).The impacts of the sustained lodging treatments, applied at 40 • (L1) and 80 • (L2) from the vertical for both varieties, were considerably greater than those arising from the transient lodging treatments (L3, L4) at identical angles when the artificial lodging treatments were induced (p < 0.05).Grain yield, harvest index, starch yield, and protein yield were significantly affected by degree of lodging and year, but the effect of their interaction was not significant (Table 1).Over 2 years, lodging treatments caused a marked drop in grain yield, harvest index, starch yield and protein yield for both varieties (Figure 4A-H

Granule Volume Distribution
Both the degree of lodging and year had significant effects on the volume proportions of A-type (>9.9 µm in diameter) and B-type (<9.9 µm) starch granules, but the effect of their interaction was not significant in this study (Table 2).Compared with un-lodged controls, the lodging treatments significantly decreased the volume proportions of starch granules sized at 0.8-2.8µm and 2.8-5.6 µm; however, they had converse effects on the size distribution of 9.9-22.8µm and 22.8-42.8µm starch granules (Table 2).The starch granules of sizes <0.8 µm and 5.6-9.9µm were not affected by lodging treatments.Overall, the treatments of L1, L2, L3, and L4 significantly reduced B-type starch granules by 9.05%, 13.07%, 6.23%, and 8.20% (mean of both cultivars), respectively, and increased A-type by 7.88%, 11.37%, 5.41%, and 7.14% (mean of both cultivars), respectively.The magnitude of changes in both cultivars arising from the sustained 80 • lodging was significantly greater than 40 • permanent lodging (p < 0.05), and the effects of transient lodging for 48 h were considerably lower in comparison to those caused by the permanent lodging.

Granule Surface Area Distribution
Lodging and year significantly influenced the surface area proportion of starch granules across various size classes, except for that with proportions of <0.8 µm (Table 3).Overall, the proportions of B-granules were decreased by different lodging treatments, although there was an increase in granules within the size range of 5.6 to 9.9 µm, and the effects of L1 and L2 permanent lodging on that were significant.Meanwhile, lodging increased the surface area proportions of A-type granules; the increases for the permanent lodging treatments of L1 and L2 were up to 11.25% and 19.05%, respectively (mean of both cultivars), and the increases for the transient lodging treatments of L3 and L4 were up to 3.53% and 6.70%, respectively (mean of both cultivars, Table 3).The findings revealed that the occurrence of lodging led to a reduction in the surface area ratios of B-type granules and a corresponding increase in that of A-type granules, and the more serious the lodging degree, the greater the effect on the changes in these proportions.

Granule Number Distribution
As depicted in Table 4, <2.8 µm and <9.9 µm starch granules made up a significant portion of the total starch granules, with their numbers accounting for 86.44-94.31%and 99.88-99.90%,respectively.This indicated that the majority of the granules examined were classified as B-type starch granules.The impacts of lodging on the numerical proportions of A-and B-type starch granules were minimal.However, lodging increased the ratio of granules <0.8 µm and 2.8-9.9 µm, and decreased the ratio of starch granules ranging from 0.8-2.8µm (Table 4), and the impacts of the sustained lodging treatments (L1, L2) for both varieties were considerably more pronounced compared to the effects of temporary lodging (L3, L4).These findings suggest that the smaller starch granules predominated in number, even though their collective contribution to the overall volume was relatively minor, and show that lodging can influence the numerical proportions of these starch granules.

Starch Viscosity Parameters
As shown in Table 5, lodging and year had significant effects on peak viscosity, hold viscosity, final viscosity, breakdown viscosity, and rebound value.Compared to the control group, treatments of L1, L2, L3, and L4 caused a drop in peak viscosity, hold viscosity, final viscosity, breakdown viscosity, and rebound value (Table 5), and the differences in those indicators all reached a significant level, except L3.The permanent and transient 40 • or 80 • lodging from the vertical caused varying degrees of reductions in these indicators.Over two years, the peak viscosity, breakdown viscosity, and rebound value differed remarkably among all lodging treatments, with a ranking of L2 < L1 < L4 < L3 < CK (Table 5, p < 0.05).Overall, treatments of L1, L2, L3, and L4 significantly reduced the peak viscosity by 9.05%, 13.07%, 6.23%, and 8.20% (mean of both cultivars), respectively, and reduced breakdown viscosity by 7.88%, 11.37%, 5.41%, and 7.14% (mean of both cultivars), respectively.The magnitude of the changes for both cultivars caused by the 80 • sustained lodging was significantly greater than that caused by the 40 • sustained lodging (p < 0.05).The impacts of sustained lodging were considerably more pronounced than those triggered by transient lodging for 48 h (p < 0.05).

Discussion
In this study, wheat crops experienced a notable decrease in grain yield due to stem lodging.We scheduled the lodging to occur at anthesis, as this is a prevalent scenario that has been observed to cause the most severe yield reductions in wheat in our region, as well as elsewhere [27,29].Even though the wheat plants in some treatments were lodged for only 48 h, relative losses of grain yield up to 18.23% and 26.25% were identified in these artificial

Discussion
In this study, wheat crops experienced a notable decrease in grain yield due to stem lodging.We scheduled the lodging to occur at anthesis, as this is a prevalent scenario that has been observed to cause the most severe yield reductions in wheat in our region, as well as elsewhere [27,29].Even though the wheat plants in some treatments were lodged for only 48 h, relative losses of grain yield up to 18.23% and 26.25% were identified in these artificial 40 • and 80 • lodging treatments, respectively.When plants with permanent stem lodging were not permitted to return to their natural growth pattern, the relative loss of grain yield grew to 41.2% and 51.1% for the 40 • and 80 • lodging treatments, respectively.Our findings are similar to previous reports in wheat [29,31,41,42], foxtail millet [43], and other cereals [32,44] under different growing conditions.However, it should be noticed that while all of these studies have concluded that lodging can lead to yield reduction, there is a discrepancy in the magnitude of yield reduction between our study and previous ones [31,35,45].This is because the timings or angles of lodging, and even the bending point where the stem is bent or broken, can vary greatly.In both cultivars, notable impacts were detected with regard to a decrease in harvest index across all lodging treatments.
One reason for our studies was that previous reports had only dealt with the effects of lodging on grain yield and yield-associated traits in wheat [26][27][28][29][30][31][32].The current research offers a precise calculation of the magnitude of reduction, granule size distribution, and viscosity parameters of starch due to lodging in wheat.In both genotypes, the reduction in starch content and yield was proportional to the severity of the lodging treatments.Artificial 80 • lodging for both genotypes led to greater reductions in starch content and yield compared to artificial 40 • lodging.The type of lodging, whether permanent or transient, is another factor that significantly affected the starch yield response to lodging.The smaller yield reductions with transient lodging at anthesis may be related to the ability of the lodged crop to stand up again.It is important to note that lodging increased grain protein content, but caused a marked drop in protein yield.Our results agree with previous studies that have stated that protein content increases in wheat can be triggered by lodging [27,46,47].Nevertheless, the extent of protein content increase in our study does not align with the aforementioned studies, a discrepancy that may be attributed to the intensity of our lodging treatments [29,48].The fact that lodging caused higher protein content than the un-lodged control treatment is consistent with the well-documented dilution effect, which describes the dilution of a finite amount of available nitrogen into more grains [27].
Starch granule size distribution, a crucial determinant of end-product quality, exhibits significant variation in response to environmental factors [49,50].The volume of A-type starch granules decreased as the plants faced lower mean temperatures, while that of B-type granules increased [51,52].Shading resulted in a considerable decrease in both the volume and surface area ratios of B-type granules [22].Waterlogging increased the volume ratio of the A-type granules [53].Our findings indicate that lodging can exert a substantial impact on both the volume and surface area ratios of A-type and B-type starch granules.Compared with the un-lodged control, lodging significantly decreased the volume ratios of granules at 0.8-5.6 µm, and resulted in contrary outcomes regarding the size distribution of 9.9-42.8µm starch granules.Additionally, lodging resulted in considerable alterations to both the volume and surface area ratio of B-type granules.The findings imply that B-type starch granules exhibited greater susceptibility to lodging treatments compared to A-type starch granules.However, the analysis revealed that lodging did not exert a significant influence on the proportions by number of A-type or B-type granules.A prior investigation into wheat starch definitively demonstrated that B-type granules originate from protrusions on A-type amyloplasts [18,54].The current findings indicate that under lodging conditions, the constrained resources for starch synthesis are predominantly allocated towards the enlargement of existing starch granules rather than the formation of more starch granules.
The gelatinization characteristics of starch are closely related to the quality of wheatbased foods, such as the texture of bread and steamed bread, the springiness and consistency of noodles, and other similar attributes [15,39,55].Additionally, the peak viscosity, trough viscosity, breakdown, final viscosity, setback, and pasting temperature of starch gelatinization play a decisive role in the application of starch [11,23].For example, studies have shown a positive correlation between the peak viscosity of starch and the quality ratings of noodles, as well as a positive relationship between the final viscosity of starch and the perceived slipperiness of noodles [4,52,56].The gelatinization characteristics of starch, especially its ability to absorb water and form a paste when heated, are influenced by the differing sizes of starch granules [1,15,57].Environmental factors can exert substantial impacts on the gelatinization properties of wheat starch [49][50][51][52][53]. Weak light after anthesis significantly reduces the peak viscosity, trough viscosity, and final viscosity [19].The gelatinization characteristics of wheat grain starch are notably modified under the stress of waterlogging and, even more so, under the combined stress of weak light and waterlogging conditions [15,53,57,58].With an increase in planting density, the peak viscosity, trough viscosity, breakdown, and final viscosity of maize starch has been shown to increase significantly [59].Our results showed that lodging caused a marked drop in peak viscosity, hold viscosity, final viscosity, breakdown viscosity, and rebound value.The results indicated that the viscosity parameter of starch could be decreased by lodging, thus affecting the stability of starch gelatinization.
Plants exposed to either sustained or episodic stress, including drought, heat, or shading, can exhibit a diverse array of intricate and fluctuating responses.Plants facing stress may have hindered capacity to generate photosynthate for the sinks [29,[60][61][62].As a result, the phase of grain development is frequently abbreviated, and the endosperm cell number is reduced, which collectively reduces the starch content in the mature grain.Various authors have documented that starch synthases, starch debranching enzymes, and starch branching enzymes play key roles in the synthesis of the granules [63,64].Stress directly impacts the activity of enzymes involved in starch biosynthesis [65,66].This diminishes the quantity, and often alters the structure of the starch granules at maturity [67].In a lodged wheat community, the typical canopy structure is compromised, photosynthetic capacity and dry matter production are decreased, and the transport of water, nutrients, or assimilates is impeded [29,36,43].It has been observed that the negative impacts of lodging on grain filling are primarily due to self-shading by leaves and panicles [30,34,68,69].We hypothesize that wheat grain has been selectively bred to accumulate starch excessively under ideal conditions, but self-shading or decreases in canopy photosynthesis due to lodging can undermine yield and quality, in part by modifying the activity of the enzymes involved in starch biosynthesis.The enzymes could be influenced at transcriptional or post-transcriptional levels in reaction to stress, leading to potentially severe impacts on crop yield and quality.Setting breeding goals or cultivation strategies will require a deep understanding of various aspects, including governance and analyzing how they function autonomously and in collaboration to regulate the process of starch synthesis to adapt to environmental extremes.Therefore, further study is required.
The distribution of granule sizes was closely correlated with starch content and viscosity parameters [4,11,16,52].Various studies have indicated that A-type granules contain 4-10% more amylase than the B-type ones [3,12,15,59].In this research, we noticed that the amylose content was positively linked to the volume ratio of granules sized 2.8-9.9 µm, and negatively linked to the ratio of 9.9-22.8µm granules.Our results align with those reported in plants under waterlogging or shading conditions [22,23], but differ from those reported in plants under different water regimes [19].The reason for these differences lies in the significant variation in the intensities of the stress treatments among the experiments.The peak viscosities or final viscosities had strongly inverse relationships with the volume ratio of midsize granules, and had a positive association with large granules [16].In the present study, we observed that the peak viscosity, trough viscosity, and rebound value of starch were positively linked to the volume ratio of 2.8-5.6 µm granules, and negatively linked to volume ratio of 9.9-22.8µm granules.A-type granules exhibited greater pasting viscosity, swelling power, and aging resistance, while B-type granules demonstrated superior gel stability [14,24,70].From the above, it is clear that lodging can reduce the volume proportion of B-type starch granules and increase that of A-type ones, which consequently diminishes the peak viscosity, trough viscosity, dilute value, and rebound value in wheat grains.

Conclusions
Lodging is one of the major constraints limiting the high yield and grain quality of wheat.This is the very first disclosure of data revealing how lodging influences the granule size distribution and viscosity parameters of starch.Through artificial sustained and transient lodging experiments spanning two years, we found that lodging caused a marked drop in both starch content and starch yield, and the relative loss of grain yield, starch yield, harvest index, and protein yield all differed remarkably across the lodging treatments, with a ranking of L2 > L1 > L4 > L3.It was also found that the relative loss of protein yield triggered by the transient lodging treatments was lower than that triggered by sustained lodging.
B-type starch granules exhibited greater susceptibility to lodging treatments compared to A-type starch granules.Lodging can reduce the volume and surface area proportions of B-type starch granules and increase those of A-type ones, and consequently, it diminishes the peak viscosity, trough viscosity, dilute value, and rebound value of wheat grains.Furthermore, we found that the smaller starch granules predominated in number, even though their collective contribution to the overall volume was relatively minor.Additionally, we also discovered that the magnitude of those changes for both cultivars arising from the sustained 80 • lodging was significantly greater than the sustained 40 • lodging, and the effects of transient lodging for 48 h were considerably lower in comparison to those caused by the sustained lodging.

Figure 2 .
Figure 2. Photographs of plots that align with the permanent 40° (A) and 80° lodging (B) of plants JM22 at anthesis.

Figure 2 .
Figure 2. Photographs of plots that align with the permanent 40 • (A) and 80 • lodging (B) of plants JM22 at anthesis.
); the permanent and transient lodging treatments of 40 • or 80 • from the vertical caused varying degrees of reduction in those indicators.The relative loss of grain yield under the L1, L2, L3, and L4 treatments were 30.22%,39.51%, 18.23%, and 26.25% (mean of two cultivars; Figure5A,B, p < 0.05), respectively; the relative loss of harvest index under the permanent lodging treatments was higher than under the transient lodging treatments for both cultivars (Figure5C,D, p < 0.05).Over two years, the relative loss of starch yield for both cultivars differed remarkably among all lodging treatments, with a ranking of L2 > L1 > L4 > L3 (Figure5E,F, p < 0.05).The relative loss of protein yield triggered by the transient lodging treatments (L3, L4) was lower than that triggered by the sustained lodging treatments (L1, L2) (Figure5G,H, p < 0.05).content; SC: starch content; PC: protein content; GY: grain yield; HI: harvest index; SY: starch yield; PY: protein yield; Y: year; C: cultivar; L: lodging.ns: not significant p > 0.05.*, **, and *** indicate significance at the 0.05, 0.01, and 0.001 levels, respectively.

Table 1 .
ANOVA of amylose content, starch content, protein content, grain yield, harvest index, starch yield, and protein yield as affected by year, cultivar, lodging, and their interaction.

Table 2 .
Effects of lodging treatments on the proportion (%) by volume of starch granules in different size classes.
• sustained lodging; L2: 80 • sustained lodging; L3: 40 • transient lodging; L4: 80 • transient lodging.Values in the same column with different letters indicate significance at the 0.05 level for the same cultivar within the same year.ns: not significant p > 0.05.** and *** indicate significance at the 0.01 and 0.001 levels, respectively.

Table 3 .
Effect of lodging treatments on the proportion (%) by surface area of starch granules in different size classes.

Table 4 .
Effect of lodging treatments on the proportion (%) by number of starch granules in different size classes.

Table 5 .
Effects of lodging treatments after anthesis on viscosity parameters of wheat starch (cP).