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\title{Effects of high temperature on rice grain development and quality
formation based on proteomics comparative analysis under field warming}



\author[1]{Wenzhe Liu}%
\author[1]{Xueqin Wang}%
\author[1]{Kailu Wang}%
\author[1]{Yingying Shen}%
\author[1]{Tongyang Yin}%
\author[1]{Yanfeng Ding}%
\author[1]{She Tang}%
\affil[1]{Nanjing Agricultural University}%


\vspace{-1em}



  
  \date{April 25, 2026}


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\selectlanguage{english}
\begin{abstract}
With the intensification of global warming, rice production is facing
new challenges. Field evidence indicates increased temperature during
rice grain-filling lead to a further deterioration of grain quality.
Clarifying the potential regulation mechanism of elevated temperature on
rice development and quality formation will be contributed to develop
suitable cultivation measures to better cope with climate warming in the
future. In this study, open field warming and DIA mass spectrometry were
conducted to explore the regulatory effects of high temperature on
pathways related to grain development and material accumulation during
the formation of rice quality. 840 differentially expressed proteins
(fold change \textgreater{} 2, p-value \textless{} 0.05) were identified
when exposed to high temperature. Among these, prolamin PPROL 14E,
PSB28, granule-bound starch synthase 1 and 26.7 kDa heat shock protein
were the most significantly regulated, and that ultimately affected the
main substances accumulation of starch and protein in the kernel, and
further degraded rice quality under high temperature. In addition, the
results provided novel targets involved in regulating the metabolism of
storage compounds under warming environment, and that will help us to
better understand the regulation mechanism of global warming on the
formation of rice quality.%
\end{abstract}%



\sloppy


\textbf{Acknowledgements:}

The authors thank Prof. Shaohua Wang for providing the seeds used in
this study, and for growing and maintaining the plants.

\textbf{Effects of high temperature on rice grain development and
quality formation based on proteomics comparative analysis under field
warming}

\textbf{Abstract:}

With the intensification of global warming, rice production is facing
new challenges. Field evidence indicates increased temperature during
rice grain-filling lead to a further deterioration of grain quality.
Clarifying the potential regulation mechanism of elevated temperature on
rice development and quality formation will be contributed to develop
suitable cultivation measures to better cope with climate warming in the
future. In this study, open field warming and DIA mass spectrometry were
conducted to explore the regulatory effects of high temperature on
pathways related to grain development and material accumulation during
the formation of rice quality. 840 differentially expressed proteins
(fold change \textgreater{} 2, p-value \textless{} 0.05) were identified
when exposed to high temperature. Among these, prolamin PPROL 14E,
PSB28, granule-bound starch synthase 1 and 26.7 kDa heat shock protein
were the most significantly regulated, and that ultimately affected the
main substances accumulation of starch and protein in the kernel, and
further degraded rice quality under high temperature. In addition, the
results provided novel targets involved in regulating the metabolism of
storage compounds under warming environment, and that will help us to
better understand the regulation mechanism of global warming on the
formation of rice quality.

\textbf{Key words:} rice; global warming; proteomics; data-independent
acquisition; grain quality

\textbf{1.Introduction}

Rice is the staple food for more than 50\% of world's population. With
the improvement of people's living standards, high-quality rice has
become more preferred by the rice production and consumption market.
However, with the rapid development of industrialization, human
activities are estimated to have caused approximately 1.0\selectlanguage{ngerman}°C (a likely
range of 0.8°C to 1.2°C) of global warming above pre-industrial levels
(IPCC, 2018). According to the fifth assessment report (AR5) completed
by the IPCC (Intergovernmental Panel on Climate Change), the global
temperature is expected to rise by 1.4-5.8°C in 2100 (IPCC, AR5, 2014).
This abnormal high temperature would seriously affect the normal growth
and development rhythm of rice and ultimately affect yield and quality
of rice (Xu et al., 2020; Jagadish, 2020). In particular, results of our
10-year field trials showed that rice quality formation generally
exhibits negative response when exposed to high temperature. The
increase of temperature leads to the significantly increased chalkiness
and decreased milling quality of rice, which could extremely reduce the
purchase expectation and the market value of rice (Dou et al., 2017).
Therefore, rice production will have to face the challenges of high
temperatures exacerbated by the intensification of climate warming in
the future.

High temperature usually affects the key processes of rice growth and
development, including germination, seedling growth, leaf emergence,
tillering, heading and maturity stage (Hakata et al., 2012; Lin et al.,
2010; Shi et al., 2016; Tsukaguchi and Iida, 2008). Among them, as the
decisive stage of rice quality formation, grain-filling is the most
sensitive period to external temperature. Grain-filling is the process
of grain development and accumulation of storage materials e.g. starch,
storage proteins and lipids, which ultimately determines the rice
quality-related indicators. An increase in temperature at this stage
would induce the decline in rice quality, including reduced milled rice
fraction, significantly increased chalky rate and chalky area (Shi et
al., 2013 Jagadish et al., 2015). As the most abundant components in
rice grain, starch had been proved to be sensitive to increased
temperature. Our previous study showed that the accumulation of total
starch and amylose in early grain-filling stage was accelerated under
the condition of increased temperature, but the accumulation speed in
later stage was significantly decreased, which resulted in the lower
content of amylose and total starch in mature grain compared to normal
temperature treatment (Tang et al., 2019). Furthermore, high temperature
during grain-filling could increase the contents of grain storage
proteins, with a significantly increased composition of glutelin and
decreased prolamin, and ultimately improved the nutritional quality of
rice. However, rice with high protein content is more susceptible to
deterioration during storage, and the appearance and eating quality of
rice could be further declined (Cao et al.,2017). In addition, high
temperature can enhance the activity of protease and accelerate the
protein transformation into soluble nitrogen compounds such as amino
acids, which would significantly increase the total amount of amino
acids and the relative content of each component in rice grain. Overall,
high temperature mainly accelerates the rate of grain-filling, but
shortens its active duration, resulting in the insufficient accumulation
of photosynthetic substances in rice grains (Kim et al., 2011; Wahid et
al., 2007). In our previous research, this change was manifested in
abnormal grain development caused by high temperature and the
accumulation balance change of starch and storage proteins, which
coordinately determines the formation of grain quality (Tang et al.,
2018; Dou et al., 2017). Although we have obtained the physiological and
biochemical evidence of high temperature in regulating grain storage
material accumulation through field trials, the regulatory mechanism
remains to be further clarified. The anabolism of rice starch and
protein is a relatively complex process, including a series of metabolic
pathways, synthesis, transport, modification, accumulation and other
processes. Therefore, we intend to conduct the investigation and
clarification of the key regulatory factors that participate in the
above process during the warming process through high-throughput
methods, which would help us to further understand the regulatory
effects of high temperature on the main pathways of rice quality
formation.

In-depth understanding of the regulation mechanism of warming on the
synthesis and metabolic pathways of grain storage materials has
important practical significance for further establishing high-quality
rice cultivation methods under climate warming. Thus, the main purpose
of this study is to further evaluate the critical period of temperature
regulation of grain-filling and to clarify the key regulatory factors
involved in grain quality formation under low-amplitude warming scene in
the actual paddy field.

\textbf{2. Materials and methods}

\textbf{2.1 Experimental site description}

The experiment was conducted at the Danyang Experimental Base of Nanjing
Agricultural University (31°56\selectlanguage{english}'39\selectlanguage{english}"N, 118deg59\selectlanguage{english}'13\selectlanguage{english}"E, 80m). The test site
belongs to the main high-yield rice cultivation area in the lower
reaches of the Yangtze River in China. The climate belongs to the
subtropical monsoon climate and the soil condition is loam with a pH
value of 6.04. The total soil nitrogen was 1.4 g\selectlanguage{english}*kg\textsuperscript{-1},
the available nitrogen was 7.8 mg\selectlanguage{english}*kg\textsuperscript{-1}, the available
phosphorus was 20.1 mg\selectlanguage{english}*kg\textsuperscript{-1} and the available
potassium was 91.7 mg \selectlanguage{english}* kg\textsuperscript{-1}. The temperature and
precipitation in the experiment are listed in the supplementary data.

\textbf{2.2 Plant materials and temperature treatment}

The high-quality rice (\emph{Oryza sativa L.} ) variety Wuyujing 3 (W3)
was used as the plant material for its sensitivity of the quality
formation to the increased temperature and the large area of cultivation
in the experimental region. In order to evaluate the individual and
co-impact effects of the increased temperature, field treatments were
conducted as follows: (i) normal temperature (CK); (ii) elevated
temperature (ET). Temperature treatment was carried out with the
free-air temperature enhancement system (FATE) from full heading (80\%
of the rice in the field starts the heading stage). Each treatment was
designed with 3 replications and 80cm intervals and protection lines
were set up between the treatment blocks to ensure the independence of
the experiment. Rice cultivation methods were adopted by seedling
raising and artificial transplanting. Field management measures
including fertilization, wet-dry alternate irrigation, prevention of
pests and weeds were carried out according to the local high-yielding
cultivation measures.

\textbf{2.3 Field warming treatment}

In order to realistically simulate the global warming scene, we have
conducted elevated temperature experiment since 2009 through the FATE
(free-air temperature enhancement) system in the rice field (Rehmani et
al., 2011\&2014; Dou et al., 2018; Tang et al., 2019). Warming treatment
was performed by FATE with 12 ceramic infrared radiator heaters
(FTE-1000-240-0-L10-Y; 1000W, 240V; 245 mm long x 60 mm wide) 1.2m above
the rice canopy in each experimental plot. Angles of the heater in the
horizontal and vertical directions are 45deg and 30deg to ensure the
consistent and stable heating of the canopy and the effective area of
infrared radiation is 1.5 x 1.5 x 3.1416 = 7.1m\textsuperscript{2},
respectively (Figure 1). Two sensors (Model HOBO U23-001) were installed
and located at the canopy height in each plot to record the temperature
and humidity changes of rice canopy. The HOBO was set to record the data
every 30 minutes, and HOBO Pro software was used for data processing.
Field meteorological data during the test period were collected from the
meteorological station (WatchDog 550) installed \textasciitilde{}100 m
away from the experimental field (Tang et al., 2019). Daytime and
nighttime of rice canopy temperature were increased by 1.568 \selectlanguage{english} and 3.089
\selectlanguage{english}, respectively (Supplemental figure 1). Temperature increase range and
trend were in accordance with the temperature range of global warming
and the rule of asymmetric warming characteristics.

\textbf{2.4 Sampling}

Rice superior spikelets (located at the upper 1/3 of the rice panicle)
flowering on the same day were tagged and sampled at 9:00 am on the 3rd,
6th, 9th, 12th and 15th day after flowering. The collected spikelets
were immediately frozen in liquid nitrogen and stored at -80 deg for
further analysis. All samples collected from the ET treatment were
within the effective warming area (Figure 1). At the mature stage, 10
holes were randomly selected from each plot, and the number of spikelets
per hole, the number of spikelets per panicle, 1000-grain weight, seed
setting rate were investigated to calculate the final yield. Harvested
spikelets were air dried under natural conditions for the determination
of starch and protein content.

\textbf{2.5 Measurement of starch and storage protein composition}

Refined rice samples were milled into flour in the liquid nitrogen, and
the starch was purified according to the instructions of Tran et al.
(2011). The starch molecules are completely dissolved by the protease,
sodium bisulfate, DMSO/LiBr (0.5\%) ethanol solution, and the proteins,
fats, and non-starch polymers are removed without starch degradation.
Starches were further debranched with isoamylase and dissolved in
DMSO/LiBr solution and physicochemical properties of starch were
identified from the milled rice. The total starches were determined by
using the protocols described in our previous study by Yang et al.
(2016).

According to the solubility of protein components in different solvents,
albumin, globulin, prolamin, and glutelin can be extracted with
distilled water, dilute hydrochloric acid, ethanol, and dilute alkali in
sequence, and the extracts are collected separately. The biuret
colorimetric method was used to determine the remaining species using
the Coomassie brilliant blue colorimetric method (Sapan et al., 1999).

\textbf{2.6 Determination of rice appearance, milling, cooking and
eating quality}

For rice appearance quality, chalk characteristics of brown rice were
observed by the cleanliness test-bed according to our previous studies
(Tang et al., 2019). Rice grains were milled using a mini universal
grinder and dried over a 200-mesh sieve. Use vernier calipers to
determine the length, width, and thickness of brown rice, and calculate
the ratio of length to width. 300 grains of brown rice were randomly
selected to detect chalky rice grains, and calculate chalky rice rate
based on the percentage of chalky rice in the total number. Chalkiness
area is determined by the proportion of chalky grain area to total grain
area.

Rice milling quality includes three indicators: brown rice rate,
polished rice rate and polished rice rate. During the maturity period,
30 rice panicles with uniform maturity were randomly harvested. After
threshing, the grains were naturally dried with the moisture content to
15\%. Brown rice percentage (BRP) and milled rice percentage (MRP) were
determined by the processing machinery SY88-TH \& SY88-TRF (Wuxi
Shanglong Grain Equipment Co., Ltd., China), respectively.

Rice cooking and eating quality was determined by the RVA-4500 (a rapid
viscometer developed by Newport scientific instruments, Australia).
Weigh 3.00g rice flour with moisture content of about 14.0\% into
aluminum box, add 25ml distilled water, and stir it up and down rapidly
for 10 times with an agitator to make the rice flour disperse evenly.
The determination of RVA characteristic parameters for rice flour was
programed as follows: the rice flour solution sample was heated at 50 \selectlanguage{english}
for 1 min, then heated to 95 \selectlanguage{english} within 3.8 min, heated at 95 \selectlanguage{english} for 2.5
min, then cooled to 50 \selectlanguage{english} within 3.8 min, and finally heated at 50 \selectlanguage{english} for
1.4 min. The characteristic parameters of RVA spectrum include: peak
viscosity (PKV), hot paste viscosity (HPV), cooling paste viscosity
(CPV), breakdown viscosity (BDV), and depletion value (setback
viscosity, SBV).

\textbf{2.6 Protein enzymatic hydrolysis}

Appropriate amount of protein sample was transferred into a 1.5ml
centrifuge tube and added with 5mm steel bead and appropriate amount of
Lysis Buffer 3, added PMSF with a final concentration of 1 mM, and EDTA
with a final concentration of 2 mM. Vortex and let stand for 5 minutes,
add DTT with a final concentration of 10 mM. Oscillate with a tissue
grinder for 2 minutes (Frequency=50HZ). 25000g centrifugation at 4 degC
for 20 minutes and add 10 mM DTT to the supernatant and water bath at 56
degC for 1 hour. After returning to room temperature, 55 mM IAM was added
and incubated in dark room for 45 minutes. Add cold acetone by 4 times
volume of the sample, and stand at -20 degC for 2h. Repeat previous step
until the supernatant is colorless. 25000g centrifugation at 4 degC for 20
minutes and discard the supernatant. Add an appropriate amount of Lysis
Buffer 3 to precipitate, followed by ultra-sonication to dissolve the
precipitated proteins. After centrifugation at 25000 g * 4 degC for 20
minutes, the supernatant was taken for quantification.

For protein extraction quality control, standard protein (0.2 \selectlanguage{greek}μ\selectlanguage{english}g / \selectlanguage{greek}μ\selectlanguage{english}l
BSA) was added sequentially to the 96-well plate A1 to A10. 0, 2, 4, 6,
8, 10, 12, 14, 16, 18\selectlanguage{greek}μ\selectlanguage{english}l, then add pure water 20, 18, 16, 14, 12, 10, 8,
6, 4, 2\selectlanguage{greek}μ\selectlanguage{english}l, add each well MAS Bright Blue G-250 Quantitative Working
Solution 180 \selectlanguage{greek}μ\selectlanguage{english}l. The OD595 was measured with a microplate reader, and a
linear standard curve was prepared based on the OD595 and protein
concentration. Dilute the protein solution to be tested several times,
add 180 \selectlanguage{greek}μ\selectlanguage{english}l of the quantitative working solution to 20 \selectlanguage{greek}μ\selectlanguage{english}l of the protein
solution and read OD595. The sample protein concentration was calculated
based on the standard curve and sample OD595. Each 10 \selectlanguage{greek}μ\selectlanguage{english}g of protein
solution was mixed with an appropriate amount of loading buffer, heated
at 95  for 5 minutes, centrifuged at 25,000 g for 5 minutes, and the
supernatant was poured into a well of a 12\% SDS polyacrylamide gel.
120V constant pressure electrophoresis for 100 minutes; After
electrophoresis, the gel was stained with Coomassie brilliant blue for 2
hours, then decolored with appropriate amount of decoloring solution
(40\% ethanol 10\% acetic acid) on the shaker every 30 minutes, in total
3\textasciitilde{}5 times.

100 \selectlanguage{greek}μ\selectlanguage{english}g of protein solution per sample and dilute with 50mM
NH\textsubscript{4}HCO\textsubscript{3} by 4 times volumes. Add 2.5 \selectlanguage{greek}μ\selectlanguage{english}g
of Trypsin enzyme in the ratio of protein: enzyme =40:1, and digest for
4 hours at 37 \selectlanguage{ngerman}°C. Add Trypsin once more in the above ratio and continue
to digest for 8 hours at 37 \selectlanguage{english}. Enzymatic peptides were desalted using a
Strata X column and vacuumed to dryness (Gillet et al., 2012; Rost et
al., 2014).

\textbf{2.7 High pH RP separation}

All samples were taken 10 ug respectively to mix, and 200 ug mixture was
diluted with 2 mL of mobile phase A (5\% ACN pH 9.8) and injected. The
Shimadzu LC-20AB HPLC system coupled with a Gemini high pH C18 column (5
um, 4.6 x 250 mm) was used. The sample was subjected to the column and
then eluted at a flow rate of 1 mL/min by gradient: 5\% mobile phase B
(95\% CAN, pH 9.8) for 10 minutes, 5\% to 35\% mobile phase B for 40
minutes, 35\% to 95\% mobile phase B for 1 minute, flow Phase B lasted 3
minutes and 5\% mobile phase B equilibrated for 10 minutes. The elution
peak was monitored at a wavelength of 214 nm and component was collected
every minute. Components were combined into a total of 10 fractions,
which were then freeze-dried.

\textbf{2.8 DDA Spectral Library and DIA analysis by nano-LC-MS/MS}

Data-Dependent Acquisition fractions and DIA samples analysis were both
performed on a Q Exactive HF X mass spectrometer (Thermo Fisher
Scientific) coupled with an Ultimate 3000 RSLCnano system (Thermo Fisher
Scientific). A nano-LC column (150 \selectlanguage{greek}μ\selectlanguage{english}m \selectlanguage{ngerman}× 30 cm, 1.8 \selectlanguage{greek}μ\selectlanguage{english}m, 100 \selectlanguage{ngerman}Å) was packed
in-house for peptide separation at a flow rate of 500 nl/min. For DDA
analysis, peptides were loaded to a C18 trap colullmn (300 \selectlanguage{greek}μ\selectlanguage{english}m \selectlanguage{ngerman}× 5 mm, 5
\selectlanguage{greek}μ\selectlanguage{english}m, Thermo Scientific) with buffer A (2\% ACN, 0.1\% FA) for 5 min, then
eluted with a gradient from 5\% to 25\% buffer B (98\% ACN, 0.1\% FA)
for 155 min, 25\% to 30\% buffer B for 10 min, and 30\% to 80\% buffer B
for 5 min. The mass spectrometry parameters were set as below: MS scan
range 350-1500 m/z; loop count 30; NCE 28; MS resolution 120 000,
maximal injection time (MIT) 50 ms; MS/MS HCD scans with resolution 30
000, MIT 100 ms; dynamic exclusion duration 30 s; isolation window 2.0
m/z; intensity threshold 2.0 e4; charge exclusion, exclude 1, 7, 8,
\textgreater{}8. For DIA analysis, the same nano-LC system and gradient
was used as DDA analysis. The DIA MS parameters were set as below: full
scan range 400-1250 m/z at resolution 120000 with MIT 50 ms; DIA
isolation window was set to 17 m/z with loop count 50 and automatic MIT,
scanned at resolution 30000; stepped NCE: 22.5, 25, 27.5; AGC target
1e6.

\textbf{2.9 Bioinformatics database}

The proteins were classfied into three categories, biological process,
cellular compartment and molecular function, by gene ontology annotation
derived from the NCBI database (www.http: //www.ebi.ac.uk/GOA/). Kyoto
Encyclopedia of Genes and Genomes (KEGG) database was used to annotate
protein pathway. Gene lists were analyzed using the STRING database to
identify the predicted interactions of proteins.

\textbf{3. Results}

\textbf{3.1 Effects of increased temperature on rice quality and
accumulation of grain storage materials}

The temperature increase of 1.6  to 3.1  during grain-filling stage
induced various changes in the accumulation of storage materials during
grain development, and further affected the rice yield and quality
indicators. Results showed that tested rice yield in warming treatment
was 21\% lower than that of the normal temperature treatment (data not
shown). Meanwhile, another significant change caused by warming is the
reduction in rice quality. Results indicated the increase in temperature
resulted in the increased grain thickness and decreased grain length.
There was no significant change in grain width, and aspect ratio of
grain shape. Compared with natural temperature, warming treatment
significantly increased rice chalky rate, chalky area and chalkiness.
Overall, the increase in temperature leads to overall deterioration in
the appearance quality of rice. In addition, elevated temperature has a
significant effect on milled rice rate and head rice rate. Compared with
the natural temperature, the milled rice rate and head rice rate were
decreased significantly. Increased temperature has led to the
deterioration of the appearance quality of rice. The increase in
temperature also caused significant changes in the rice cooking quality
indicators. Gelatinization characteristics peak viscosity (PKV),
hot-paste viscosity (HPV), and gelatinization temperature (GT) were
significant increased and the final viscosity was decreased (Table 1).

Starch and storage proteins are the main substances that constitute the
grain storage material. Under elevated temperature conditions, grain
total starch was increased significantly, of which amylopectin was
significant increased when responded to high temperature, while amylose
decreased significantly compared with normal temperature treatment, and
that further led to changes in the proportion of amylopectin /amylose.
The response of grain storage protein components to high temperature
appears as a significant increase in glutelin and a significant decrease
in prolamin. There results suggested high temperature during
grain-filling stage has a general negative impact on rice quality
parameters.

\textbf{3.2 Quantitative expression of rice grain proteins under
elevated temperature}

In order to further explore the regulation mechanism of the effects of
increased temperature on rice quality, we conducted the dynamic
proteomic analysis of the rice grains in the early stage of grain
filling and a total of 23968 unique peptides and 5872 unique proteins
(Supplementary Table 1). The expression and annotation proteins
identified in each period were listed and Pearson correlation analysis
showed the repeatability of these protein samples was above 97\%.
Furthermore, we enriched all proteins with COG and GO to perform
functional analysis, and results showed that the effect of elevated
temperature was mainly in regulating the translation, post-translational
modification, protein conversion and signal transduction during the
grain-filling stage.

Differentially expressed proteins (DEPs) were defined as proteins with a
FOLD CHANGE [?]2 and p VALUE \textless{} 0.05. In the present study, DEPs
were identified at least twice in three biological replicates and had
the same change trend. Based on these criteria, 112 DEPs were found in
ET-3d (3d after heading under elevated temperature treatment) and CK-3d
(3d after heading under normal temperature treatment) groups, of which
66 were upregulated and 46 were downregulated (Figure 2). In ET-6d and
CK-6d treatments, 118 DEPs were identified, of which 51 were up-regulated
and 67 were down-regulated. Comparing to the CK-9d, 65 proteins were
up-regulated and 201 proteins were down-regulated in the ET-9d. In
ET-12d and CK-12d treatments, 144 DEPs were identified, of which 59 were
up-regulated and 85 were down-regulated. In addition, 200 DEPs were
found during the 15d after heading, including 30 up-regulated and 170
down-regulated proteins. The volcano plots and protein annotation of
DEPs in ET and CK (3d, 6d, 9d, 12d and 15d) treatments are shown in
Figure 3 and Supplementary Table 2. According to the GO enrichment
analysis, the prominent GO terms for CC enriched by five stages were the
cell, cell part, organelle. Identified proteins were predominantly
distributed in metabolic process, cellular process and single-organism
process. Based on the molecular function, the DEPs were mainly
classified into catalytic activity, binding, transporter activity and
structural molecule activity (HT vs CK 3d, 6d, 9d 12d). The top GO MF
categories that were enriched by HT-6d and CK-6d DEPs, including the
catalytic activity, binding, enzyme regulator activity and transporter
activity (Figure 4).

Differentially expressed proteins were further classified into five
stages with KEGG pathway. Differentially metabolic process were defined
as pathway with p VALUE \textless{} 0.05. The proteome of both the
treatments revealed changes in major metabolic pathways. The major
metabolic pathways in HT-3d and CK-3d were photosynthesis-antenna
proteins, metabolism of xenobiotics by cytochrome P450, drug
metabolism-cytochrome P450, photosynthesis, axon guidance, retinol
metabolism (Figure 5). In HT-6d and CK-6d, the main pathways were
homologous recombination, AMPK signaling pathway, inositol phosphate
metabolism, plant hormone signal transduction, NF-kappa B signaling
pathway, ether lipid metabolism, MAPK signaling pathway-plant. Among
these, the metabolic pathways enriched in HT-9d and CK-9d mainly include
mannose type O-glycan biosynthesis, porphyrin and chlorophyll
metabolism, tryptophan metabolism, ABC transporters, phenylpropanoid
biosynthesis, isoflavonoid biosynthesis, other types of O-glycan
biosynthesis, limonene and pinene degradation. Pathway enrichment
analysis of HT-12d and CK-12d DEPs identified significantly enriched in
ribosome, mitophagy-yeast, homologous recombination, C5-Branched dibasic
acid metabolism. Moreover, two major metabolic pathways, fructose and
mannose metabolism, and indole alkaloid biosynthesis were identified in
HT-15d and CK-15d. Results suggested that temperature had significantly
different regulatory effects on different stages of grain development.

\textbf{3.3 Identification of differentially expressed proteins related
to rice development and quality formation}

A total of 748 unique proteins were identified based on their specific
expressions when exposed to high temperature. After removing 302
proteins with lower scores and unknown functional classification, 39
proteins related to rice development and quality formation were
distinguished (Table 2, Figure 6). In this study, the expression of 16.9
kDa class I heat shock protein in rice grains was significantly reduced
3 days after heating, which was down-regulated by 84\% compared with CK
treatment. However, the expression of heat shock factor binding protein
1 involved in heat shock protein synthesis was significantly increased.
Furthermore, the expression levels of HSP70s at early grain-filling
stages were only about half of normal treatment (0.47, 0.5, and 0.43
times reduction at 3d, 6d 9d after flowering, respectively). Results
showed the most significant change was the 26.7 kDa heat shock protein
of the sHSPs family, and its expression was increased significantly at
6, 9, and 12 days after flowering, with 6.78, 3.06, and 5.44 folds
compared to the CK treatment. At the 12th day after flowering, the
expressions of 18.0kda class II heat shock protein, 24.1kda heat shock
protein and heat shock protein 82 were up-regulated to varying degrees
(2.26, 2.01, 3.63 folds respectively) when subjected to high
temperatures.

Expression of proteins related to rice quality formation were also
regulated under field warming conditions. Expressions of glutelin
anabolism proteins were regulated at the beginning of grain-filling. For
example, the glutelin type-A 3, glutelin type-B 1, glutelin type-B
5-like expression levels of rice grains were significantly up-regulated
by 2.79, 2.49, and 3.3 folds, respectively. At the same time, the
expression levels of glutelin type-B 1-like and glutelin type-B 2-like
proteins were decreased significantly. Furthermore, the expression
levels of globulin 19 kDa globulin, globulin 1s allele and basic 7S
globulin were increased significantly, which is consistent with the
increase of globulin content measured in mature grains. In this study,
the significant decrease in prolamin under warming conditions was mainly
at the 12th day after flowering and expression analysis of prolamin
PPROL 14E and prolamin PPROL 14E-like proteins showed that they were
both decreased significantly (0.33, 0.38 folds) at this stage comparted
to the normal temperature treatment. As the main component of rice
grains, the accumulation of rice grain starch has been proved to be
sensitive to elevated temperature. The synthesis of rice grain starch is
mainly regulated by a series of protein families, including SSS, SBE,
DBE, GBSS. In this study, the GBSS was down-regulated at the 6th day
after flowering under increased temperature. While proteins related to
amylopectin synthesis obtained no significant changes when compared to
the CK. The expression levels of granule-bound starch synthase at 6d, 9d
and 12d after flowering were significantly lower than that of the
control and the expression levels of soluble starch synthase responsible
for the synthesis of amylopectin SS4 and SSS2-3 were decreased under
high temperatures.

Results of this study showed that another key growth and development
process regulated by the increased temperature during grain-filling
stage was the photosynthetic system of rice plant. In this study, the
expression levels of the chlorophyll a-b binding protein 1B-21,
chlorophyll a-b binding protein P4, and chlorophyll a-b binding protein
7 in chlorophyll ab binding protein were significantly up-regulated at
the beginning of grain-filling (2.22, 2.03, 3.3 times), when compared to
the CK. On the other hand, the expression of PSB28, which is responsible
for water splitting, had a downward trend through the grain-filling
period, and reaching a significant level at 12 days after
flowering.~Furthermore, protein TIC 62 (translocon at the inner envelope
membrane of chloroplasts) responsible for the dynamic balance of the
proteome was significantly downregulated at 9d after flowering.
Photosynthesis is the process by which light energy is converted into
chemical energy and stored, and thus it is essential for the
accumulation of rice grain assimilation.

\textbf{4. Discussion}

\textbf{4.1 Assessment of field warming and the impact on rice quality
formation}

Since 2009, we have continuously carried out warming experiments in rice
fields based on the open-field free air temperature enhancement system
(FATE), and the effects of increased temperature on rice growth and
development were guardedly studied. The ultimate goal of our study is to
find key regulatory factors or metabolic pathways involved in rice
growth process and apply appropriate field cultivation measures to
reasonably respond to the challenge of climate warming on rice. The FATE
device is suspended above the field and uses 12 sets of ceramic infrared
heaters to perform uniform heating in an area of
7.1m\textsuperscript{2}. In the fully activated state, the daytime
canopy temperature of rice can be increased by 2.4 on the basis of
natural temperature, and the rice canopy night temperature can be
increased by 5.4. This warming range is within the prediction of
possible temperature increase by 1.4-5.8 \selectlanguage{ngerman}°C at the end of the 21st
century by IPCC (IPCC, AR5, 2014). Furthermore, the increase in night
temperature is significantly greater than that during the day and that
is consistent with the asymmetric trend of climate warming (Pachauri et
al., 2014). Compared with closed or semi-closed warming scenario, the
warming method and effect adopted in this project could closely simulate
climate warming characteristics and that provides a more reliable
platform for us to conduct related experiments in the actual field.

Rice quality is a complex characteristic, including appearance, milling,
nutrition, cooking and eating quality. Our field evidence shows that the
overall temperature increase has a relatively negative effect on rice
quality, including significantly increased chalky rate, chalky area and
chalkiness. Meanwhile, the milling quality indicators milled rice rate
and head rice rate were decreased significantly, and that would
exceedingly reduce the market recognition of rice. Our previous
researches have been devoted to exploring the mechanism of the influence
of increased temperature on rice quality and to our knowledge, the
changes of external temperature inevitably affect the morphological
composition and structure of the grain storage material, and that
further induce the changes of related quality traits (Dou et al., 2017;
Dou et al., 2018; Tang et al., 2018; Tang et al., 2019). Among these
attributes, eating and cooking quality (ECQ) is one of the most
important indicators, especially from the consumer's perspective. The
eating quality refers to the sensory perception of consumers on rice,
and is related to the gloss, flavor, and viscosity of rice. Although the
physical and chemical properties of starch in rice endosperm can be used
as an indirect indicator of ECQ, it is still a difficult task to assess
ECQ through these traits. At the same time, the increase of glutelin
content in rice grain is particularly obvious under the condition of
increased temperature, which leads to a change in the overall balance of
grain storage materials and has a negative impact on the taste and
appearance quality of the rice.

\textbf{4.2 Overview of DIA quantitative proteomics analysis}

Due to the complex mechanism of rice quality formation, this study
conducted the proteomics analysis on physiological pathways such as
starch synthesis and metabolism, storage protein accumulation, and plant
photosynthesis under warming conditions, and to identify the key
regulation factors in pathways related to temperature response. In
recent years, proteomics-based mass spectrometry has made significant
progress from sample preparation to liquid chromatography and instrument
detection, making it possible to identify more specific expressed
proteins in cells or tissues with excellent accuracy and repeatability
(Tsou et al., 2015). Data independent acquisition (DIA) is widely used
in proteomics analysis due to its higher protein coverage rate and
reliable data acquisition ability (Renaud \& Sumarah, 2016, Searle et
al., 2015). Compared with iTRAQ, the advantage of DIA technology is that
it can effectively measure protein molecules with extremely low
abundance in complex samples, which greatly improves the reliability of
quantitative analysis and has high quantitative accuracy and
repeatability. In this study, samples of interest went through mass
spectrometry data collection in data dependent acquisition (DDA) mode.
MaxQuant was then used to carry out database search identification
process and obtain all detectable non-redundant high-quality MS/MS
spectral information as DIA spectral library, which contains fragment
ion intensity and retention time describing the peak characteristics of
the peptide, for quantification. Here, we identified 23968 unique
peptides and 5872 unique proteins, which could be specifically regulated
by the increased temperature during rice grain filling. Those DEPs
coordinate and execute their biological behaviors based on their
metabolic pathways. Therefore, KEGG pathway-based analysis would be
contributed to further understand their biological functions. From our
results, these identified specifically expressed proteins have large
differences in temporal and spatial characteristics, which provided the
obstacles to our further identification and screening of key regulators.
Therefore, returning to the essential relationship between grain-filling
and quality formation, we further screened the key proteins that we
believe are specifically regulated by warming during the quality
formation process from the perspectives of plant photosynthesis, grain
starch and storage protein accumulations.

\textbf{4.3 Key regulatory factors contributed to rice quality formation
under elevated temperature}

Photosynthesis is the process by which light energy is converted into
chemical energy and stored, and it is also the source of accumulation of
rice grain assimilation. Chlorophyll content and metabolic enzyme
activity are closely related to the strength of photosynthesis. In our
case, chlorophyll a-b binding protein 1B-21, chlorophyll a-b binding
protein P4, and chlorophyll a-b binding protein 7 were significantly
up-regulated, which induced the acceleration of the synthesis and
binding of chlorophyll (Ballottari et al., 2012). Meanwhile, the
expression levels of photosystem I reaction center subunit VI and
oxygen-evolving enhancer protein 3 from the photosystem I were also
increased significantly under warming conditions, and that may explain
the accelerated grain filling rate during the early grain-filling stage
induced by elevated temperature, and the significant increase in the
accumulation rate of grain materials compared with the normal
temperature treatment. However, the expression of PSB28, which is
responsible for water splitting, had a downward trend throughout the
period, obtaining a significant low level at 12d after flowering under
elevated temperature (0.4 folds). That may inhibit electron transfer and
weakens signal transmission, thereby weakening photochemical reactions
and resulting in decreased cell chlorophyll and photosynthesis (Wada et
al., 2019). Previous research shows that the optical system II (PS II)
is the most sensitive element to temperature in the electron
transmission chain (Zhang et al., 2011). It would be interesting to
further investigate whether PSB28 could be the most critical component
affected by high temperature during the photosynthesis process.

To our knowledge, the contents and ratio of starch and storage protein
in rice grains are the decisive factors in determining the final rice
quality. Rice starch synthesis is regulated by various enzymes,
including SSS, SBE, DBE and GBSS. GBSS is the main enzyme responsible
for amylose synthesis. Wx protein encoded by the Waxy gene GBSS-I can
tightly bind to the starch granules and promote the synthesis of
amylose. High temperatures can lead to downregulation of gene expression
that regulates GBSS synthesis, resulting in decreased amylose content
and increased amylopectin content (Dian et al. 2005; Fujita et al.
2006). Research by Denver (1996) shows that GBSS is not only related to
the synthesis of amylose, but also to the extension of amylopectin in
starch granules. However, the exact effect of GBSS on the extension of
normal starch granules is still unclear. Our results showed that the
GBSS enzyme was down-regulated at 6d after flowering under elevated
temperature. However, enzymes related to amylopectin synthesis did not
change significantly. From 6d to 12d after flowering, the expression
level of granule-bound starch synthase was significantly lower than that
of the control. Under high temperature, the amylose content of mature
rice grains was significantly lower than that of CK treatment, while the
amylopectin content was significantly increased (Ahmed et al., 2015).
Expression levels of the soluble starch synthase SS4 and SSS2-3,
responsible for the synthesis of amylopectin, were also decreased under
high temperatures (Yamakawa, 2012). This change may reduce the
activities of granular starch synthase and soluble starch synthase, and
lead to change in the ratio of amylose and amylopectin, which eventually
affected the physical and chemical properties of starches in rice grain
(Tang et al., 2019).

Rice storage proteins include albumin, globulin, glutelin and prolamin.
Prolamin is directly deposited in the endoplasmic reticulum cavity in
the form of intracellular protein particles, and finally buds from the
endoplasmic reticulum in the form of spherical protein bodies (PBIs).
While glutelin is efficiently converted into mature form by vacuolar
processing enzymes, and forms irregular protein bodies II (PBII)
together with \selectlanguage{greek}α-\selectlanguage{english}globulin (Krishnan et al., 1992; Kumamaru et al., 2010).
The results of this study showed that warming had significant up- or
down-regulation effects on the expression of storage protein
family-related regulatory factors at different periods. For example, the
expression of glutelin type-A and type-B proteins were either
significantly up-regulated or down-regulated at 3d and 6d after
flowering, and there is no obvious rule for the regulation mode of these
regulatory factors under warming conditions. Based on our understanding
of glutelin, its synthesis pathway is still unclear, and the presence of
many unknown glutelin genes increases the difficulty in understanding
the expression pattern under warming condition. Therefore, this study
has not been able to essentially find the direct reasons for the changes
in the final grain storage protein content.

However, several types of protein species (ribosomal protein species,
superfamily II DNA and RNA helicase, and molecular chaperone IbpA)
related to biosynthesis and processing of proteins was found to be
affected by high temperature. Ribosomes are the primary sites for
protein synthesis, and different species of ribosomal proteins play an
essential role in translation, ribosome structure, and biogenesis in
protein anabolism (Moin et al., 2016). In our study, the ribosomal
protein species (25S, 30S, 40S, 50S and 60S) exhibited significant
decreases during the middle stage of grain-filling, which may cause the
reduction in the protein biosynthesis and maintain the balance between
synthesis and degradation of proteins (Moin et al., 2016). The reduction
in protein content related to translation, such as RNA recognition motif
(RRM) domains, eukaryotic initiation factors (eIFs) and elongation
factors (EFs), indicates the adverse effects of high temperature on rice
protein synthesis. Furthermore, a series of molecular chaperone heat
shock proteins (Hsps) were identified to be significantly up-regulated
when exposed to high temperature. Heat shock proteins Heat shock protein
is a highly conserved peptide in structure and could be activated and
produced in large quantities when plants are subjected to abiotic stress
(Timperio et al., 2008). In this study, the two most sensitive heat
shock proteins are HSP70 and 26.7 kDa heat shock protein of the sHSPs (s

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