It is well established that SS1, SS2a, and SS3a are responsible for AP chain elongation, while SBE1 and SBE2 control the formation of branched structures in AP (Pandey et al., 2012). The chain length distribution or degrees of polymerization (DP) in AP shows very important effects on rice quality and starch physicochemical properties (Buléon et al., 1998). The activity of rice SS1 is higher than that of SS2a and SS3a in rice endosperm. SS1 preferentially synthesizes short chains of DP 6–12. In the ss1 mutant, chains of DP 8–12 are decreased, whereas DP 6–7 chains are increased, which indicates that SS1 elongates DP 6–7 chains to DP 8–12 chains of AP (Fujita et al., 2006; Li et al., 2018). SS3a is another important enzyme for AP synthesis, and the activity of SS3a is higher than that of SS2a but lower than that of SS1. SS3a is mainly responsible for the generation of long chains (DP ≥ 30) in AP (Fujita et al., 2007). The ss3a mutant showed significantly reduced long chains of AP and abnormal starch granule morphology, which results in a floury endosperm (Ryoo et al., 2007). This result suggested that long chains catalyzed by SS3a are critical for maintaining normal structures of starch granules. In contrast, no obvious starch granule or morphological defects were observed in ss1 seeds (Fujita et al., 2006). However, the ss1/ss3a double mutant of japonica rice is sterile (Fujita et al., 2011; Hanashiro et al., 2011). These data indicated that the reduction of short chains in AP might not be enough for morphological alteration of starch granules, while the simultaneous reduction of both short and long chains could affect the formation of starch granules. SS2a was proposed to mainly produce intermediate chains (DP 13–25) of AP (Umemoto et al., 2002). The activity of SS2a is significantly different between indica and japonica. SS2a from japonica might be an inactive allele showing no or very low activity in vitro, while the indica allele has relatively higher activity (Umemoto et al., 2004). SS2a is a key gene that mainly determines rice GT, an important physiochemical property for rice eating and cooking quality (Gao et al., 2003, 2011). Introducing the indica SS2a allele into japonica rice could convert the structure of AP from the S-type (mainly in japonica cultivars) to the L-type (mostly in indica cultivars) and increase GT significantly as well (Nakamura et al., 2005).

It was generally believed that SSs (SS1, SS2a, and SS3a) were only involved in AP synthesis. Recent studies noted that these SSs might also affect AM synthesis. The short chains of AP produced by SSs could supply substrates for the synthesis of AM (Zhu et al., 2020). SS1 is a dominant enzyme for AP synthesis, especially for the short chain of DP 6–12, as its activity accounts for approximately 70% of the total SS activity (Fujita et al., 2006). Thus, deficiency of SS1 would cause a great reduction in AP. However, the appearance of seeds and starch granules remained normal, and the AC remained unchanged in the ss1 mutant (Fujita et al., 2006). Moreover, sbe2 mutant seeds present a higher AC than wild-type seeds (Butardo et al., 2011), and knockdown of SS1 in sbe2 results in AC compensation (Abe et al., 2014). We deduced that the increase of AC in total starch was due to greatly impaired AP synthesis in sbe2, whereas defects in AM biosynthesis subtly balanced the ratio of AM to AP and returned the AC to WT level in the double mutant generated from leaky mutant of SS1crossed with sbe2 mutant. These results strongly suggested that SS1 plays important roles not only in AP synthesis but also in AM synthesis, and the short chains of AP (DP 6–12), which are mainly produced by SS1, might be important substrates for AM synthesis.

SS2a might be another SS gene involved in AM synthesis. Introducing the high activity allele SS2a^Ind (indica allele of SS2a) into rice plants could raise the AC whereas the effects are ecotype dependent (Yang et al., 2018; You et al., 2020). In the Wx^a background, SS2a^Ind could increase AC dramatically (Tian et al., 2009), while in the Wx^b background, SS2a^Ind has a minor effect on AC (Zhang et al., 2020b). The total activity of the GBSSI protein generated by Wx^a is higher than that generated by Wx^b (Wang et al., 1995). Therefore, we deduced that the intermediate chains of AP (DP 13–25) produced by SS2a might be substrates for AM synthesis, and Wx^a may use these substrates with higher efficiency than Wx^b. It will be very interesting to investigate the AC alteration in which SS2a^Ind is introduced into the genetic background with weaker Wx alleles, such as Wx^mq and Wx^hp. SS3a might not be involved in the synthesis of AM. Loss of function of SS3a caused a significant reduction in long chains (DP > 30) in AP and no obvious alteration in AM synthesis (Fujita et al., 2007), although the relative ratio of AM to AP was increased and the AC was increased in the ss3a mutant. These results indicated that long chains of AP produced by SS3a might not be used as substrates in AM synthesis. Thus, we proposed that short and intermediate chains (DP < 25) of AP might be important substrates for AM synthesis and both SS1and SS2a play important roles in this process. More evidence for this conception should be collected in the future by using other technologies, such as radio isotope tracer.

SBE1 and SBE2 show different enzyme activities and biological functions in starch synthesis. SBE1 presents higher activity than SBE2 in rice endosperm. SBE2 has a high affinity for AP, while SBE1 is involved in branch addition in both AP and AM (Nakamura et al., 2010). Although no significant morphological defects were found in sbe1 seeds, both intermediate chains of DP 12–21 and long chains of DP ≥ 37 were reduced which resulted in a GT decrease in sbe1 seeds (Satoh et al., 2003). SBE2 seems to play a more important role in AP synthesis than SBE1 (Zhu et al., 2012; Nakata et al., 2018; Baysal et al., 2020). Short chains (DP < 17) were decreased greatly, and opacity or chalkiness appearance occurred in sbe2 (ae, amylose extender) seeds (Nishi et al., 2001; Butardo et al., 2011). This indicated that SBE2, similar to SS1, is very important for short-chain synthesis. Interestingly, in the ss1/sbe2 (ss1/ae) double mutant, similar to ss1/ss3a, normal starch granules could not be formed, and very few seeds could be produced (Abe et al., 2014). These results suggested that AP synthesis and the chain length distribution are very important for starch granule formation, which eventually affects rice yield and quality.

As we mentioned above, amylose synthesis is mainly controlled by the Wx gene, and many allelic Wxs, such as Wx^a, Wx^b, Wxⁱⁿ, Wx^mq, Wx^lv, and wx, explain a major AC variation in rice germplasm. With the development of biotechnology, more novel Wx alleles were generated and many important rice materials with different AC were produced by genetic modification or gene editing recently, and most of them occurred at coding and promoter region of Wx gene (Liu et al., 2014; Zeng et al., 2020; Huang et al., 2020a; Xu et al., 2021). Moreover, the Wx gene can be finely regulated at the transcriptional, post-transcriptional, and translational levels, and the factors involved in these processes are also important for AC modulation and rice quality control (Figure 1A).

Many transcription factors have been reported to transactivate Wx expression by binding to cis-elements, such as 31 bp core sequences (Ge et al., 2000) and Em boxes (Cheng et al., 2002) at the upstream of Wx. For example, a bHLH transcription factor, OsBP5, together with its interacting protein, OsEBP89, binds to 31 bp and synergistically regulates the transcription of Wx (Zhu et al., 2003). OsbZIP58, a key factor in starch synthesis, was verified to directly bind to cis-elements from both Wx and SBEI and coordinately control AP and AM biosynthesis at the transcriptional level (Wang et al., 2013). Knockout or knockdown of these genes would cause alterations in AC and rice quality.

Several dull genes were isolated recently, and some of them were found to regulate Wx expression at the post-transcriptional level. Du1 encodes an mRNA splicing factor and participates in the splicing of the first intron in rice Wx (Zeng et al., 2007). Du3 encodes a protein similar to nuclear cap binding protein subunit 2 and is involved in the nuclear export of Wx mRNA (Isshiki et al., 2008). The splicing efficiency of the Wx gene was reduced and the AC decreased significantly in both du1 and du3 mutants, which indicated that post-transcriptional regulation of Wx is very important for rice quality control. In addition to the genes that regulate Wx expression and GBSSI activity, an increasing number of novel factors and pathways have been revealed to influence AM synthesis. PTST1 (Protein Targeting to Starch), which is responsible for AM synthesis in Arabidopsis leaves, was newly found to help the GBSS protein localize to starch (Seung et al., 2015). The CBM48 domain at the C-terminus of PTST1 is important for its binding activity to starch. Although GBSS itself has weak binding activity to starch, it could be recruited by PTST1 through the coiled-coil domain and subsequently bound to starch mediated by the CBM48 domain in PTST1 (Seung, 2020). OsGBP, a homolog of PTST1 in rice, could interact with both rice GBSS genes, Wx and GBSS2, in vitro. However, only AM biosynthesis in leaves but not in endosperm was greatly impaired in the osgbp mutant (Wang et al., 2020). Therefore, OsGBP may mainly function in chloroplasts, and there might be other factors involved in GBSSI locating starch in the endosperm. FLO6 (floury endosperm), another homolog of PTST1 in rice, is the most likely such gene. FLO6 contains the CBM48 domain at the C-terminus and can interact with GBSSI, GBSSII, and ISA1 to help them target starch in endosperm. Total starch and AC decreased significantly, and starch granules were abnormal in the flo6 mutant (Peng et al., 2014; Zhang et al., 2021). FLO6 might be involved in AM synthesis in rice. The transcription factor OsNF-YB1 can bind to a G-box in the Wx promoter and activate its expression (Xu et al., 2016). Moreover, OsNF-YB1 can interact with several transcription factors, such as OsNF-YC12 and OsbHLH144, and form a complex to regulate starch synthesis genes, including Wx and ISA1 (Bello et al., 2019). Mutants of osnf-yb1, osnf-yc12, and osbhlh144 displayed similar phenotypes to flo6, such as chalky endosperm, reduced grain weight, and decreased total starch and AC. Moreover, OsNF-YC12 binds to the promoter of FLO6 and directly regulates its expression (Xiong et al., 2019). Further study to reveal the biological function of FLO6 and OsGBP and their regulatory network in GBSSI activity modification will be very meaningful for elucidating starch synthesis and quality control in rice seeds.

In addition to flo6, many new flo mutants with floury endosperm were isolated, and most of them showed abnormal starch granules and reduced grain weight and AC (Figure 1B). Many FLO genes have been cloned and found to engage in different biological processes. FLO10, FLO14, and FLO18 encode pentatricopeptide repeat (PPR) proteins involved in RNA binding and metabolism in plant mitochondria. The processing of mitochondrial NAD genes, such as NAD1 and NAD5, was defective in flo10, flo14, or flo18 mutants (Wu et al., 2019; Xue et al., 2019; Yu et al., 2020a). NAD genes encode subunits of NADH dehydrogenase that are essential for ATP production and mitochondrial development. FLO13, known as OsNDUFA9, encodes subunit mitochondrial complex I. Loss of OsNDUFA9 changes the mitochondrial structure and greatly impairs the development of rice endosperm (Hu et al., 2018). FLO16, known as OsCMDH, encodes an NAD-dependent cytosolic malate dehydrogenase. ATP and AC were obviously reduced in the flo16 mutant (Teng et al., 2019). These reports suggested that regulators involved in the NADH pathway are essential for both mitochondrial morphogenesis and endosperm development in rice. Moreover, FLO2 was predicted to encode a tetratricopeptide repeat (TPR) domain-containing protein (She et al., 2010). The candidate gene responsible for FLO4 encodes a pyruvate orthophosphate dikinase (Kang et al., 2005; Zhang et al., 2018b). OsHsp70 is the gene responsible for the FLO11 phenotype, and FLO15 encodes glyoxalase I (Zhu et al., 2018; You et al., 2019). Clarifying the biological function of these FLO genes will be beneficial to uncover new components and pathways influencing seed development and rice quality in the future.

The early development of endosperm has a great influence on the quality and yield of rice. Polycomb repressive complex 2 (PRC2), which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), is essential for the early development of endosperm (Figure 1C; Tonosaki and Kinoshita, 2015). Fertilization-independent endosperm (FIE) is an important component of PRC2. There are two FIE genes, OsFIE1 and OsFIE2, in the rice genome (Luo et al., 2009). Seed defect phenotypes, such as limited endosperm development, semisterile spikelets, and impaired grain size and quality, were obviously displayed in osfie (osfie1 or osfie2) mutants (Nallamilli et al., 2013; Li et al., 2014; Huang et al., 2016; Cheng et al., 2020). Some MADS-box genes, which are mainly responsible for floral organ identity, seem to be involved in the process of PRC2-mediated early endosperm development. For example, OsMADS6 plays an essential role in endosperm nutrient accumulation. In the osmads6 mutant, starch filling was blocked, and the relative contents of protein and soluble sugar increased, which resulted in altered grain size and quality (Yu et al., 2020b). ChIP-PCR analysis revealed that H3K27 is trimethylated in vegetative tissues where OsMADS6 is silenced (Zhang et al., 2010). Other type II MADS box genes, such as OsMADS1 (Liu et al., 2018; Yu et al., 2018), OsMADS34 (Ren et al., 2016), and OsMADS29 (Yin and Xue, 2012; Nayar et al., 2013), also contribute to early endosperm development and might be regulated by PRC2. In rice lacking OsEMF2B, another important component of PRC2, the expression of the above MADS-box genes was altered (Conrad et al., 2014; Xie et al., 2015). Moreover, several type I MADS-box genes, such as OsMADS78, OsMADS79, OsMADS87, and OsMADS89, also played essential roles in early seed development (Paul et al., 2020). OsMADS78 and OsMADS79 could interact with OsMADS87 and OsMADS89 and form a heterodimerized complex. Transgenic seeds deficient in these type I MADS-box genes exhibited accelerated endosperm cellularization and altered grain quality. The expression of these MADS genes was negatively correlated with OsFIE1 (Folsom et al., 2014). All these data suggested that PRC2-MADS might be an essential cascade for early seed development. As an increasing number of MADS genes have been found to be highly expressed in endosperm, we speculate that numerous MADS genes will function in PRC2-mediated early endosperm development and grain quality control in the future.

The reduction of the AC at HT is mostly caused by the downregulation of the Wx gene (Larkin and Park, 1999). Compared to the transcriptional inhibition of Wx by HT, post-transcriptional regulation induced by HT seems more important, especially in the Wx^b background. A single nucleotide polymorphism (SNP, G to T) at the splicing site of the first intron in Wx^b causes low splicing efficiency (Cai et al., 1998; Isshiki et al., 1998). The splicing efficiency is temperature-dependent. The splicing efficiency and mature transcripts of Wx^b under cool temperature conditions (18°C) were much higher than those under optimal temperature conditions (25°C) and HT conditions (33°C). Two major mature transcripts could be generated from the Wx^b allele under optimal temperature conditions. The large one is spliced after CT repeats (site 2), and the small one is spliced near the donor site of Wx^a (site 1; Zhang et al., 2014). Two transcripts are generated almost equally from Wx^b under optimal temperature conditions, while the large transcript represents the majority under HT, and the small transcript mainly exists at cool temperature (Larkin and Park, 1999; Zhang et al., 2014). These results suggested that the selection of donor sites in alternative splicing of Wx^b is temperature-dependent. Alternative splicing at site 1 was suppressed by HT but promoted by cool temperature (Figure 2). We deduced that some important factors might control the selection of splicing sites and that the activity of these factors is sensitive to temperature.

Indica rice is usually more tolerant to HT than japonica in terms of AC. Under the same Wx^b background, the drop in AC in 9311 (indica) under HT was much smaller than that in Nipponbare (japonica). Using the CSSLs between 9311 and Nipponbare, several QTLs, qHAC8a, qHAC8b, and qHAC4, responsible for AC stabilization under HT were characterized. Introducing the indica allele of these loci into Nipponbare could enhance the splicing efficiency of Wx^b, which suggested that increasing the pre-mRNA processing efficiency of the Wx gene might be an important regulatory mechanism for maintaining AC stability at HT (Figure 3A; Zhang et al., 2014). The results from MADS7-RNAi plants strongly supported this hypothesis. The floral identity gene OsMADS7 was mildly expressed in endosperm but strongly induced by HT. Suppression of OsMADS7 could improve the stability of rice AC under HT. Dynamic qRT-PCR revealed that both the expression level and the pre-mRNA processing efficiency of the Wx gene were enhanced in OsMADS7 RNAi seeds under HT during almost the entire filling stage. OsMADS7 might be the gene that can negatively regulate the expression or alternative splicing of the Wx gene under HT (Zhang et al., 2018a). Moreover, dynamic analysis revealed that grain filling rate is higher at HT than that in optimal temperature condition in both wild type ZH11 and MADS7-RNAi seeds. However, the difference in grain filling rate between HT and optimal temperature condition is smaller in MADS7-RNAi than that in ZH11, which might be another reason for relatively stable AC in MADS7-RNAi under HT condition (Zhang et al., 2018a).

In addition to Wx, the expression of many other starch biosynthesis genes was also changed at HT during the filling stage in rice. Overall, SS2a and SS3a were slightly downregulated, and SS1 was induced by HT. SBE1 changed slightly, whereas SBE2 decreased significantly under HT (Yamakawa et al., 2007; Liao et al., 2015). Alterations in the expression of these genes are also important for rice quality control under HT. SS3a and SBE2 are critical genes for AP synthesis. Significant downregulation of SBE2 and SS3a indicated that AP synthesis might be impaired by HT (Zhao et al., 2020). However, AM synthesis should be much more impaired than AP synthesis, since AC was reduced greatly under HT (Lin et al., 2020). The relative abilities of AM Vs AP biosynthesis under HT might be lower than those under optimal temperature conditions. As mentioned above, SS1 or SS2a is responsible for both AM and AP synthesis. Considering the great reduction in GBSSI activity (Zhang et al., 2014), the high expression of SS1 at HT might be more beneficial to the synthesis of AP than that of AM, so the reduction in AC at HT should be explained in part by the high expression of SS1. Similar to that of SS1, the high activity of the indica allele of SS2a might make AC more sensitive to HT. In contrast, knocking down SS2a might be beneficial to improve the quality of japonica rice under HT because it not only reduces rice GT but also diminishes AC effects caused by HT.

The PRC2-MADS pathway might also be essential for the regulation of rice endosperm development under HT conditions. Genome-wide association analysis revealed that one of the PRC2 components, OsFIE1, is a major locus for grain size regulation under HT conditions (Dhatt et al., 2021). The expression of OsFIE1 in endosperm can be suppressed by heat stress. Seed development in the osfie1 background was more sensitive to HT than that in the WT. OsMADS87 was negatively regulated by OsFIE1 but induced by HT. OsMADS87 RNAi seeds were more tolerant to HT than WT seeds by using the alteration of seed size as a trait (Figure 3C; Chen et al., 2016).

Some FLO genes might also be responsible for seed development under HT. For example, the expression of FLO2 in response to HT was different between cultivars, which indicated that FLO2 may be involved in heat tolerance during the grain filling stage (She et al., 2010). FLO11 encodes the heat shock protein OsHsp70-2, whose expression was sensitive to HT. More chalky grains were generated in the flo11 mutant than in the WT when the rice was grown at 28°C but not at 24°C, indicating that FLO11 may function under elevated temperature at the milky stage (Tabassum et al., 2020). HSPs (heat shock proteins) are molecular chaperones that delay irreversible aggregation of denatured proteins under HT condition or other stress. The expression of HSP is regulated by HSF (heat shock transcription factors) whose activity was affected by Ca²⁺ sensor calmodulin (CaM) in plants (Wu et al., 2012; Bourgine and Guihur, 2021). Transcripts of several HSP genes could be induced by HT (Sarkar et al., 2009), which suggested that many HSPs, HSFs and CaMs in the Ca²⁺–dependent heat shock signaling pathway might be essential for acquired thermotolerance of rice quality.

Dynamic analysis of gene expression in the rice endosperm revealed that α-amylases, such as Amy1A, Amy3A, and Amy3E, were greatly induced by HT. Knocking down these α-amylases significantly improved rice appearance quality under HT (Figure 3B; Hakata et al., 2012), which suggested that α-amylases might play key roles in the formation of grain quality under HT. The induced expression of starch-hydrolyzing α-amylases implied that a high speed of starch degradation might be another important cause of the increased grain chalkiness under HT conditions. The transcription factor OsbZIP58 might be an essential regulator of α-amylases. Knocking out OsbZIP58, the expression of Amy1A, Amy3A, Amy3E, and Amy1C could be increased, and osbzip58 mutants produced more chalky grains than WT at HT (Xu et al., 2020). It seems that OsbZIP58 is an effective suppressor of α-amylases in rice endosperm. It might be beneficial to increase the expression of OsbZIP58 under HT to improve the appearance quality of rice.