The conventional starch from cereals is usually high in energy and easily digested, absorbed and converted into blood sugar in human small intestine, while resistant starch (RS) is hardly enzymatically hydrolysed into glucose in human small intestine and only fermented into beneficial short-chain fatty acids in large intestine (Jukanti et al., 2020; Shen et al., 2022). Thus, RS-rich foods can not only effectively reduce the glycaemic index, increase satiety and prevent blood sugar-related diseases but also contribute to the prevention of intestinal-related diseases by improving the intestinal micro-environment and decreasing colonic pH (Zaman and Sarbini, 2016). Rice is an important staple-food crop and a major source of starch for majority of the population, especially in Asia. However, the RS content in conventionally cultivated rice is usually <1%, which is far below the daily recommended RS intake for humans (Yadav et al., 2010). Therefore, breeding rice rich in RS is an important target for rice variety improvement.

Rice endosperm is the chemical reservoir storing more than 80% starch with varied amounts of amylose and amylopectin, which is mainly controlled by several key starch synthase-related genes (SSRGs; Huang et al., 2021). Currently, there have been a few successful cases of altering the composition and structure of starch to enhance RS content in rice endosperm by modifying the expression of two kinds of SSRGs, SBE and SS3/SSIII, encoding starch branching enzyme and soluble starch synthase III, respectively (Butardo Jr. et al., 2012; Guo et al., 2020; Miura et al., 2022; Zhou et al., 2016; Zhu et al., 2012). Loss of function of SBE3/SBEIIb resulted in increased RS in rice endosperm, while there is no significant effect of SBE1/SBEI mutation on RS level, but the sbe1sbe3 double mutation could further enhance RS content substantially based on sbe3 single mutation, indicating functional redundancy of these two isoforms in RS formation (Zhu et al., 2012). In rice, there are two isoforms of SS3/SSIII, SS3a/SSIIIa/SSIII-2 and SS3b/SSIIIb/SSIII-1 (Huang et al., 2021). Zhou et al. (2016) reported that the ssIIIa/ss3a loss-of-function mutant had a high RS content in cooked rice. However, the role and genetic effects of most other SSRGs, such as SS3b, in the formation of RS are not clear.

In rice, SS3a and SS3b, two genes encoding the SS3 isoforms, and their gene structure, protein domain and amino acid sequence were quite conserved (Figure S1a–c; Table S2). SS3b was expressed in low abundance in developing rice grains, but its expression trend was consistent with that of SS3a (Figure S1d,e). It implied that SS3b may also have a similar function as SS3a in regulating starch synthesis and RS formation in rice endosperm. Thus, we created SS3a and SS3b mutants by CRISPR/Cas9 genome editing technology in the japonica rice cultivar Nipponbare (WT). After several generations of genotype and phenotype screening, we obtained several homozygous mutant lines, including SS3a single mutants (ss3a), SS3b single mutants (ss3b) and their pyramiding double mutant lines (ss3a-ss3b) (Figure 1a,b). There was no significant changes in the seedling growth and plant development of all edited rice lines compared with their wild type (WT; Figure S2a,b).

The digestive characteristics of the grains from the above mutants were firstly determined. The results showed that the RS content in ss3a mutants increased significantly to 4.76%–5.01% while that in WT was only 0.58% (Figure 1c). Moreover, the total digestible starch (TDS) content and digestion rate in ss3a mutants decreased significantly (Figure 1d,e), which was consistent with previous report (Zhou et al., 2016). In ss3b mutants, there was no significant change in the contents of RS and TDS as well as digestion rate compared with those in wild type. But SS3b mutation could significantly enhance the RS level in the background of SS3a mutation, resulting in the RS content reaching 9.54%–9.73% in the ss3a-ss3b double mutants, and further decreased TDS content and digestion rate (Figure 1d,e).

Except digestion properties, the SS3b mutation had no significant effect on other grain physicochemical properties, could further strengthen the change of grain physicochemical phenotype combined with the SS3a mutation (Figure 1f,g and Figure S3a–c; Tables S3 and S4). These results suggested that SS3b can only have a significant effect on starch synthesis and RS formation in rice endosperm based on SS3a mutation, indicating ss3b mutant has synergistic effects on ss3a mutant in increasing RS content.

The starch granule morphology of ss3a and ss3a-ss3b mutants was obviously abnormal, showing most compound starch grains composed of only a few starch granules, instead, dozens of starch granules formed a compound starch grain in the wild type (Huang et al., 2021), and some starch granules were spherical (Figure 1i–l and Figure S3e–h). Furthermore, the starch granules in each compound starch granule complex were closely linked, but loosely arranged between the adjacent compound starch granules in ss3a and ss3a-ss3b mutants (Figure 1i–l). These results explained the high chalkiness phenotype in ss3a and ss3a-ss3b mutants. The starch granule morphology of ss3b mutant had no obvious change compared with the wild type.

As to starch fine structure, the true amylose fraction (Am) increased significantly and the proportion of amylopectin long chains (Ap2) decreased dramatically in ss3a and ss3a-ss3b mutants, and the range of changes in ss3a-ss3b mutants was larger than that in ss3a mutants, but no significant change occurred in ss3b mutants (Figure S5a). Besides, the amylopectin B₂ chains with degree of polymerization (DP) 22–37 increased in ss3a mutants, while the amylopectin A chains with DP 6–9 and B₁ chains with DP 13–21 decreased dramatically in ss3a and ss3a-ss3b mutants (Figure 1q). The X-ray diffraction (XRD) results showed that the starch relative crystallinity of ss3a and ss3a-ss3b mutants was significantly reduced (Figure S3d). Correspondingly, the area ratio of the characteristic peak, which representing the amylose–lipid complex as well as the type 5 RS (RS5), was significantly increased in the ss3a and ss3a-ss3b mutants, reaching 1.7 and 2.3 times that of the wild type, respectively, consistent with the changes in triglyceride and RS contents (Figure 1h). Expectedly, the SS3b mutation had no significant effect on the relative crystallinity and RS5 content of rice endosperm starch compared with the wild type (Figure 1h and Figure S3d). It implied that the increased contents of amylose and amylose–lipid complex are the main reasons for the high RS content in ss3a and ss3a-ss3b mutants.

For transitory starch in leaves, there was no significant change in starch composition, structure, and starch granule morphology after SS3a mutation, while in ss3b mutants, TSC and proportions of amylopectin B chains (DP > 12) were significantly decreased, AAC and true AC were slightly increased, and starch granules were irregular in shape and rough in surface, similar to the effect of SS3a mutation on endosperm starch synthesis (Figure 1m–r, Figures S4 and S5b). The TSC and proportions of amylopectin B chains in leaves of ss3a-ss3b mutant were further decreased, and the abnormal morphology of starch granules was aggravated, but there was no significant change on AC (Figure 1m–r, Figures S4 and S5b). These results suggested that SS3b plays a major role in leaf starch synthesis and ss3a mutant has synergistic effects on ss3b mutant.

In conclusion, our data indicated that SS3a and SS3b have synergistic effects in starch biosynthesis. In rice endosperm, further mutation of SS3b could strengthen the effect of SS3a mutation on alteration of starch physicochemical properties and RS content. A new rice germplasm with significantly increased RS content and improved digestive properties was created by co-knockout SS3a and SS3b. These results provide a new strategy to breed novel rice varieties with high RS content and further help to elucidate the functions of SSRGs in cereals.

传统谷物淀粉通常能量较高，容易在人体小肠中被消化吸收并转化为血糖，而抗性淀粉（RS）在人体小肠中很难酶解成葡萄糖，只能发酵成有益的短链脂肪大肠中的酸（Jukanti等人，  2020；Shen等人，  2022）。因此，富含RS的食物不仅可以有效降低血糖指数、增加饱腹感、预防血糖相关疾病，而且还可以通过改善肠道微环境、降低结肠pH值，有助于预防肠道相关疾病（Zaman和Sarbini） ，  2016）。稻米是一种重要的主粮作物，也是大多数人口（尤其是亚洲）淀粉的主要来源。然而，传统栽培稻中的RS含量通常<1%，远低于人类每日推荐的RS摄入量（Yadav等，  2010）。因此，选育富含RS的水稻是水稻品种改良的重要目标。

水稻胚乳是储存80%以上淀粉的化学储库，其中含有不同量的直链淀粉和支链淀粉，主要受几个关键淀粉合酶相关基因控制(SSRGs; Huang et al .,  2021 )。目前，通过改变编码淀粉分支酶和可溶性淀粉合酶III的两种SSRGs SBE和SS3 / SSIII的表达来改变淀粉的组成和结构以提高水稻胚乳中RS含量的成功案例。分别（Butardo Jr.等，  2012；Guo等，  2020；Miura等，  2022；Zhou等，  2016；Zhu等，  2012）。SBE3/SBEIIb功能丧失导致水稻胚乳中RS含量增加，而SBE1/SBEI突变对RS水平没有显着影响，但sbe1sbe3双突变可以在sbe3单突变的基础上进一步大幅提高RS含量，表明功能冗余RS 形成中这两种同工型的差异（Zhu et al .,  2012）。在水稻中，SS3/SSIII 有两种同工型：SS3a/SSIIIa/SSIII-2 和 SS3b/SSIIIb/SSIII-1（Huang等，  2021）。周等人。 ( 2016 ) 报道ssIIIa/ss3a功能丧失突变体在米饭中具有较高的 RS 含量。然而，大多数其他 SSRG（例如SS3b）在 RS 形成中的作用和遗传效应尚不清楚。

在水稻中，SS3a和SS3b是编码 SS3 亚型的两个基因，其基因结构、蛋白结构域和氨基酸序列相当保守（图 S1a-c；表 S2）。SS3b在发育中的水稻籽粒中低丰度表达，但其表达趋势与SS3a一致（图S1d，e）。这表明SS3b在调节水稻胚乳淀粉合成和RS形成方面可能也具有与SS3a相似的功能。因此，我们通过CRISPR/Cas9基因组编辑技术在粳稻品种日本晴（WT）中创建了SS3a和SS3b突变体。经过几代基因型和表型筛选，我们获得了多个纯合突变体系，包括SS3a单突变体（ss3a）、SS3b单突变体（ss3b）及其金字塔双突变体系（ss3a - ss3b）（图1a，b）。与野生型相比，所有编辑的水稻品系的幼苗生长和植物发育没有显着变化（WT；图S2a，b）。

首先测定了上述突变体的谷粒的消化特性。结果显示，ss3a突变体中的RS含量显着增加至4.76%–5.01%，而WT中的RS含量仅为0.58%（图1c）。此外， ss3a突变体的总可消化淀粉（TDS）含量和消化率显着下降（图1d，e），这与之前的报道一致（Zhou et al .,  2016）。ss3b突变体中RS和TDS含量以及消化率与野生型相比没有显着变化。但SS3b突变可以显着提高SS3a突变背景下的RS水平，导致ss3a - ss3b双突变体中RS含量达到9.54%–9.73%，并进一步降低TDS含量和消化率（图1d，e）。

除消化特性外，SS3b突变对籽粒其他理化特性没有显着影响，与SS3a突变结合可进一步加强籽粒理化表型的变化（图1f，g和图S3a-c；表S3和S4）。这些结果表明，SS3b只能在SS3a突变的基础上对水稻胚乳中淀粉合成和RS形成产生显着影响，表明ss3b突变体对ss3a突变体在增加RS含量方面具有协同作用。

ss3a和ss3a - ss3b突变体的淀粉粒形态明显异常，大多数复合淀粉粒仅由少数淀粉粒组成，而野生型则由数十个淀粉粒形成复合淀粉粒(Huang et al .,  2021 ) ），一些淀粉颗粒是球形的（图1i-l和图S3e-h）。此外，在ss3a和ss3a - ss3b突变体中，每个复合淀粉颗粒复合体中的淀粉颗粒紧密相连，但在相邻复合淀粉颗粒之间排列松散（图 1i-l）。这些结果解释了ss3a和ss3a - ss3b突变体中的高白垩性表型。ss3b突变体的淀粉粒形态与野生型相比没有明显变化。

淀粉精细结构方面， ss3a和ss3a - ss3b突变体中真直链淀粉比例（Am）显着增加，支链淀粉长链比例（Ap2）显着下降，且ss3a-ss3b突变体变化幅度大于ss3a - ss3b突变体。ss3a突变体，但ss3b突变体没有发生显着变化（图 S5a）。此外，在ss3a突变体中，聚合度（DP）22-37的支链淀粉B ₂链增加，而DP 6-9的支链淀粉A链和DP 13-21的B _1链在ss3a和ss3a - ss3b突变体中急剧减少。（图 1q）。 X射线衍射（XRD）结果表明，ss3a和ss3a - ss3b突变体的淀粉相对结晶度显着降低（图S3d）。相应地，代表直链淀粉-脂质复合物以及5型RS（RS5）的特征峰的面积比在ss3a和ss3a - ss3b突变体中显着增加，达到野生型的1.7和2.3倍，分别与甘油三酯和RS含量的变化一致（图1h）。预期，与野生型相比， SS3b突变对水稻胚乳淀粉的相对结晶度和RS5含量没有显着影响（图1h和图S3d）。这表明直链淀粉和直链淀粉-脂质复合物含量的增加是ss3a和ss3a - ss3b突变体中RS含量高的主要原因。

对于叶片中的瞬时淀粉， SS3a突变后淀粉组成、结构和淀粉颗粒形态没有明显变化，而在ss3b突变体中，TSC和支链淀粉B链的比例（DP>12）显着降低，AAC和true AC略有增加，淀粉颗粒形状不规则，表面粗糙，类似于SS3a突变对胚乳淀粉合成的影响（图1m-r，图S4和S5b）。ss3a - ss3b突变体叶片中TSC和支链淀粉B链比例进一步降低，淀粉颗粒形态异常加剧，但AC无明显变化（图1m-r，图S4和S5b）。这些结果表明SS3b在叶淀粉合成中起主要作用，并且ss3a突变体对ss3b突变体具有协同作用。

总之，我们的数据表明SS3a和SS3b在淀粉生物合成中具有协同作用。在水稻胚乳中， SS3b的进一步突变可以增强SS3a突变对淀粉理化性质和RS含量改变的影响。通过共敲除SS3a和SS3b，创建了一种新的水稻种质，其 RS 含量显着增加，消化特性得到改善。这些结果为培育高RS含量水稻新品种提供了新策略，并进一步有助于阐明SSRGs在谷物中的功能。