MFP1 defines the subchloroplast location of starch granule initiation.

Starch is one of the major carbohydrate storage compounds in plants. The biogenesis of starch granules starts with the formation of initials, which subsequently expand into granules. Several coiled-coil domain-containing proteins have been previously implicated with the initiation process, but the mechanisms by which they act remain largely elusive. Here, we demonstrate that one of these proteins, the thylakoid-associated MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1), specifically determines the subchloroplast location of initial formation. The expression of MFP1 variants "mis"-targeted to specific locations within chloroplasts in Arabidopsis results in distinctive shifts in not only how many but also where starch granules are formed. Importantly, "re" localizing MFP1 to the stromal face of the chloroplast's inner envelope is sufficient to generate starch granules in this aberrant position. These findings provide compelling evidence that a single protein MFP1 possesses the capacity to direct the initiation and biosynthesis machinery of starch granules.

Arabidopsis thaliana B-GATA factors repress starch synthesis and gravitropic growth responses.

Plants perceive the direction of gravity during skotomorphogenic growth, and of gravity and light during photomorphogenic growth. Gravity perception occurs through the sedimentation of starch granules in shoot endodermal and root columella cells. In this study, we demonstrate that the Arabidopsis thaliana GATA factors GNC (GATA, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED) and GNL/CGA1 (GNC-LIKE/CYTOKININ-RESPONSIVE GATA1) repress starch granule growth and amyloplast differentiation in endodermal cells. In our comprehensive study, we analysed gravitropic responses in the shoot, root and hypocotyl. We performed an RNA-seq analysis, used advanced microscopy techniques to examine starch granule size, number and morphology and quantified transitory starch degradation patterns. Using transmission electron microscopy, we examined amyloplast development. Our results indicate that the altered gravitropic responses in hypocotyls, shoots and roots of gnc gnl mutants and GNL overexpressors are due to the differential accumulation of starch granules observed in the GATA genotypes. At the whole-plant level, GNC and GNL play a more complex role in starch synthesis, degradation and starch granule initiation. Our findings suggest that the light-regulated GNC and GNL help balance phototropic and gravitropic growth responses after the transition from skotomorphogenesis to photomorphogenesis by repressing the growth of starch granules.

Further insight into the involvement of PII1 in starch granule initiation in Arabidopsis leaf chloroplasts.

The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis leaves, SS4 is the main enzyme that control starch granule initiation, but in its absence, SS3 partly fulfills this function. How these proteins collectively act to control the initiation of starch granules remains elusive. PII1 and SS4 physically interact, and PII1 is required for SS4 to be fully active. However, Arabidopsis mutants lacking SS4 or PII1 still accumulate starch granules. Combining pii1 KO mutation with either ss3 or ss4 KO mutations provide new insights of how the remaining starch granules are synthesized. The ss3 pii1 line still accumulates starch, while the phenotype of ss4 pii1 is stronger than that of ss4. Our results indicate first that SS4 initiates starch granule synthesis in the absence of PII1 albeit being limited to one large lenticular granule per plastid. Second, that if in the absence of SS4, SS3 is able to initiate starch granules with low efficiency, this ability is further reduced with the additional absence of PII1.

Increasing amyloplast size in wheat endosperm through mutation of PARC6 affects starch granule morphology.

The determination of starch granule morphology in plants is poorly understood. The amyloplasts of wheat endosperm contain large discoid A-type granules and small spherical B-type granules. To study the influence of amyloplast structure on these distinct morphological types, we isolated a mutant in durum wheat (Triticum turgidum) defective in the plastid division protein PARC6, which had giant plastids in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A- and B-type granules than those of the wild-type. The mutant had increased A- and B-type granule size in mature grains, and its A-type granules had a highly aberrant, lobed surface. This morphological defect was already evident at early stages of grain development and occurred without alterations in polymer structure and composition. Plant growth and grain size, number and starch content were not affected in the mutants despite the large plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, did not increase plastid or starch granule size. We suggest TtPARC6 can complement disrupted TtARC6 function by interacting with PDV2, the outer plastid envelope protein that typically interacts with ARC6 to promote plastid division. We therefore reveal an important role of amyloplast structure in starch granule morphogenesis in wheat.

Starch granule size: Does it matter?

Nature has developed starch granules varying in size from less than 1 µm to more than 100 µm. The granule size is an important factor affecting the functional properties and the applicability of starch for food and non-food applications. Within the same botanical species, the range of starch granule size can be up to sevenfold. This review critically evaluated the biological and environmental factors affecting the size of starch granules, the methods for the separation of starch granules and the measurement of size distribution. Further, the structure at different length scales and properties of starch-based on the granule size is elucidated by specifying the typical applications of granules with varying sizes. An amylopectin cluster model showing the arrangement of amylopectin from inside toward the granule surface is proposed with the hypothesis that the steric hindrance for the growth of lamellar structure may limit the size of starch granules.

Characterization of Oil Body and Starch Granule Dynamics in Developing Seeds of Brassica napus.

Brassica napus is the most important oilseed crop in the world, and the lipid was stored in the oil body (OB) in the form of triacylglycerol. At present, most of studies on the relationship between oil body morphology and seed oil content in B. napus was focused on mature seeds. In the present study, the OBs in different developing seeds of B. napus with relatively high oil content (HOC) of about 50% and low oil content (LOC) of about 39% were analyzed. It was revealed that the size of OBs was first increased and then decreased in both materials. And in late seed developmental stages, the average OB size of rapeseed with HOC was higher than that of LOC, while it was reversed in the early seed developmental stages. No significant difference was observed on starch granule (SG) size in HOC and LOC rapeseed. Further results indicated that the expression of genes that involved in malonyl-CoA metabolism, fatty acid carbon chain extension, lipid metabolism, and starch synthesis in the rapeseed with HOC was higher than that of rapeseed with LOC. These results give some new insight for understanding the dynamics of OBs and SGs in embryos of B. napus.

Effects of the duration of post-silking drought on the starch physicochemical properties of waxy maize.

BACKGROUND: Drought is a major abiotic stress that affects the physicochemical properties of cereal starch. However, quantitative information on the effects of drought duration on the starch quality of waxy maize, a special maize-type starch composed of nearly pure amylopectin, has been lacking. The effects of post-silking drought duration 1-10 (DS10), 1-20 (DS20), and 1-30 (DS30) days after pollination on the physicochemical properties of starch were assessed from 2019 to 2020 using two waxy maize hybrids as materials. RESULTS: With extending drought duration, the starch granule size and average amylopectin chain length of Jingkenuo2000 (JKN2000) gradually increased, with those of Suyunuo5 (SYN5) being the highest for DS20, followed by DS30. All drought durations decreased the degree of branching of both hybrids, with the lowest value obtained for DS30 and DS20 in JKN2000 and SYN5, respectively. Relative crystallinity increased for DS30 in both hybrids but its responses for DS10 and DS20 differed. Pasting viscosities and gelatinization enthalpy were decreased and retrogradation percentage was increased by drought stress. The lowest pasting viscosities were observed for DS30, and the highest retrogradation percentage was found for DS10 in general. CONCLUSION: Post-silking drought led to the pasting and retrogradation properties deteriorating, with decreased pasting viscosities and increased retrogradation percentage. The decrease in viscosity was caused by enlarged granules. Meanwhile, the increased proportion of amylopectin chains with a degree of polymerization of 25-36 resulted in lower viscosity and higher retrogradation. © 2022 Society of Chemical Industry.

Starch granule size and shape characterization of yam (Dioscorea alata L.) flour using automated image analysis.

BACKGROUND: Roots, tubers and bananas (RTB) play an essential role as staple foods, particularly in Africa. Consumer acceptance for RTB products relies strongly on the functional properties of, which may be affected by the size and shape of its granules. Classically, these are characterized either using manual measurements on microscopic photographs of starch colored with iodine, or using a laser light-scattering granulometer (LLSG). While the former is tedious and only allows the analysis of a small number of granules, the latter only provides limited information on the shape of the starch granule. RESULTS: In this study, an open-source solution was developed allowing the automated measurement of the characteristic parameters of the size and shape of yam starch granules by applying thresholding and object identification on microscopic photographs. A random forest (RF) model was used to predict the starch granule shape class. This analysis pipeline was successfully applied to a yam diversity panel of 47 genotypes, leading to the characterization of more than 205 000 starch granules. Comparison between the classical and automated method shows a very strong correlation (R2 = 0.99) and an absence of bias for granule size. The RF model predicted shape class with an accuracy of 83%. With heritability equal to 0.85, the median projected area of the granules varied from 381 to 1115 µm2 and their observed shapes were ellipsoidal, polyhedral, round and triangular. CONCLUSION: The results obtained in this study show that the proposed open-source pipeline offers an accurate, robust and discriminating solution for medium-throughput phenotyping of yam starch granule size distribution and shape classification. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Starch granule initiation in Arabidopsis thaliana chloroplasts.

The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.

Starch synthases SSIIa and GBSSI control starch structure but do not determine starch granule morphology in the absence of SSIIIa and SSIVb.

KEY MESSAGE: High levels of two major starch synthases, SSIIa and GBSSI, in ss3a ss4b double mutant rice alter the starch structure but fail to recover the polygonal starch granule morphology. The endosperm starch granule is polygonal in wild-type rice but spherical in double mutant japonica rice lacking genes encoding two of the five major Starch synthase (SS) isozymes expressed in endosperm, SSIIIa and SSIVb. Japonica rice naturally has low levels of SSIIa and Granule-bound SSI (GBSSI). Therefore, introduction of active SSIIa allele and/or high-expressing GBSSI allele from indica rice into the japonica rice mutant lacking SS isozymes can help elucidate the compensatory roles of SS isozymes in starch biosynthesis. In this study, we crossed the ss3a ss4a double mutant japonica rice with the indica rice to generate three new rice lines with high and/or low SSIIa and GBSSI levels, and examined their starch structure, physicochemical properties, and levels of other starch biosynthetic enzymes. Lines with high SSIIa levels showed more SSI and SSIIa bound to starch granule, reduced levels of short amylopectin chains (7 ≤ DP ≤ 12), increased levels of amylopectin chains with DP > 13, and consequently higher gelatinization temperature. Lines with high GBSSI levels showed an increase in amylose content. The ADP-glucose content of the crude extract was high in lines with low or high SSIIa and low GBSSI levels, but was low in lines with high GBSSI. Addition of high SSIIa and GBSSI altered the starch structure and physicochemical properties but did not affect the starch granule morphology, confirming that SSIIIa and SSIVb are key enzymes affecting starch granule morphology in rice. The relationship among SS isozymes and its effect on the amount of substrate (ADP-glucose) is discussed.

The CBM48 domain-containing protein FLO6 regulates starch synthesis by interacting with SSIVb and GBSS in rice.

KEY MESSAGE: FLO6 is involved in starch synthesis by interacting with SSIVb and GBSS in rice. Starch synthesized and stored in plastids including chloroplasts and amyloplasts plays a vital role in plant growth and provides the major energy for human diet. However, the molecular mechanisms by which regulate starch synthesis remain largely unknown. In this study, we identified and characterized a rice floury endosperm mutant M39, which exhibited defective starch granule formation in pericarp and endosperm, accompanied by the decreased starch content and amylose content. The abnormal starch accumulation in M39 pollen grains caused a significant decrease in plant fertility. Chloroplasts in M39 leaves contained no or only one large starch granule. Positional cloning combined with complementary experiment demonstrated that the mutant phenotypes were restored by the FLOURY ENDOSPERM6 (FLO6). FLO6 was generally expressed in various tissues, including leaf, anther and developing endosperm. FLO6 is a chloroplast and amyloplast-localized protein that is able to bind to starch by its carbohydrate-binding module 48 (CBM48) domain. Interestingly, we found that FLO6 interacted with starch synthase IVb (SSIVb) and granule-bound starch synthase (GBSSI and GBSSII). Together, our results suggested that FLO6 plays a critical role in starch synthesis through cooperating with several starch synthesis enzymes throughout plant growth and development.

Anatomical study on the developmental process of the swollen internodes of Phryma (Phrymaceae, eudicots).

MAIN CONCLUSION: We discovered that the internodal swellings of Phryma (eudicots) stems were same as the internodal pulvini of Poaceae (monocots) from the viewpoints of internal structures and functions. The stems of eudicots are usually rod-shaped and are composed of nodes, attached by leaves, and internodes. The internodes of some species, belonging to the clade 'asterids' and its sister clade 'Caryophyllales' of eudicots, have swellings, which have negative tropism, at the basal or apical part of each internode. To know the internal features of the swollen internodes, we performed outer morphological and anatomical studies on the swollen internodes of Phryma, eudicots, one of the genera having swollen internodes, from the winter bud stage to the flowering stage. The results revealed the following: (i) the swollen regions of the internodes were composed of less lignified tissues (e.g., endodermis without Casparian strips, and xylem having less lignified xylem fibers); (ii) the internodal less lignified parts were supported by collenchyma; (iii) the endodermis includes amyloplasts, having accumulated starch granules, which would function as statoliths for negative gravitropism. Consequently, we determined that the swollen parts of the Phryma internodes are same as the internodal pulvini of Poaceae of monocots from the viewpoints of internal structures and functions.

Loss of PROTEIN TARGETING TO STARCH 2 has variable effects on starch synthesis across organs and species.

Recent work has identified several proteins involved in starch granule initiation, the first step of starch synthesis. However, the degree of conservation in the granule initiation process remains poorly understood, especially among grass species differing in patterns of carbohydrate turnover in leaves, and granule morphology in the endosperm. We therefore compared mutant phenotypes of Hordeum vulgare (barley), Triticum turgidum (durum wheat), and Brachypodium distachyon defective in PROTEIN TARGETING TO STARCH 2 (PTST2), a key granule initiation protein. We report striking differences across species and organs. Loss of PTST2 from leaves resulted in fewer, larger starch granules per chloroplast and normal starch content in wheat, fewer granules per chloroplast and lower starch content in barley, and almost complete loss of starch in Brachypodium. The loss of starch in Brachypodium leaves was accompanied by high levels of ADP-glucose and detrimental effects on growth and physiology. Additionally, we found that loss of PTST2 increased granule initiation in Brachypodium amyloplasts, resulting in abnormal compound granule formation throughout the seed. These findings suggest that the importance of PTST2 varies greatly with the genetic and developmental background and inform the extent to which the gene can be targeted to improve starch in crops.

The modulatory roles and regulatory strategy of starch in the textural and rehydration attributes of dried noodle products.

Noodles are popular staple foods globally, and dried noodle products (DNPs) have gained increasing attention due to recent changes in consumer diet behavior. Rapid rehydration and excellent texture quality are the two major demands consumers make of dried noodle products. Unfortunately, these two qualities conflict with each other: the rapid rehydration of DNPs generally requires a loose structure, which is disadvantageous for good texture qualities. This contradiction limits further development of the noodle industry, and overcoming this limitation remains challenging. Starch is the major component of noodles, and it has two main roles in DNPs. It serves as a skeleton for the noodle in gel networks form or acts as a noodle network filler in granule form. In this review, we comprehensively investigate the different roles of starch in DNPs, and propose strategies for balancing the conflicts between texture and rehydration qualities of DNPs by regulating the gel network and granule structure of starch. Current strategies in regulating the gel network mainly focused on the hydrogen bond strength, the orientation degree, and the porosity; while regulating granule structure was generally performed by adjusting the integrity and the gelatinization degree of starch. This review assists in the production of instant dried noodle products with desired qualities, and provides insights into promising enhancements in the quality of starch-based products by manipulating starch structure.

Disruption of LLM9428/OsCATC Represses Starch Metabolism and Confers Enhanced Blast Resistance in Rice.

Catalases (CATs) are important self-originating enzymes and are involved in many of the biological functions of plants. Multiple forms of CATs suggest their versatile role in lesion mimic mutants (LMMs), H2O2 homeostasis and abiotic and biotic stress tolerance. In the current study, we identified a large lesion mimic mutant9428 (llm9428) from Ethyl-methane-sulfonate (EMS) mutagenized population. The llm9428 showed a typical phenotype of LMMs including decreased agronomic yield traits. The histochemical assays showed decreased cell viability and increased reactive oxygen species (ROS) in the leaves of llm9428 compared to its wild type (WT). The llm9428 showed enhanced blast disease resistance and increased relative expression of pathogenesis-related (PR) genes. Studies of the sub-cellular structure of the leaf and quantification of starch contents revealed a significant decrease in starch granule formation in llm9428. Genetic analysis revealed a single nucleotide change (C > T) that altered an amino acid (Ala > Val) in the candidate gene (Os03g0131200) encoding a CATALASE C in llm9428. CRISPR-Cas9 targetted knockout lines of LLM9428/OsCATC showed the phenotype of LMMs and reduced starch metabolism. Taken together, the current study results revealed a novel role of OsCATC in starch metabolism in addition to validating previously studied functions of CATs.

Three Diverse Granule Preparation Methods for Proteomic Analysis of Mature Rice (Oryza sativa L.) Starch Grain.

Starch is the primary form of reserve carbohydrate storage in plants. Rice (Oryza sativa L.) is a monocot whose reserve starch is organized into compounded structures within the amyloplast, rather than a simple starch grain (SG). The mechanism governing the assembly of the compound SG from polyhedral granules in apposition, however, remains unknown. To further characterize the proteome associated with these compounded structures, three distinct methods of starch granule preparation (dispersion, microsieve, and flotation) were performed. Phase separation of peptides (aqueous trypsin-shaving and isopropanol solubilization of residual peptides) isolated starch granule-associated proteins (SGAPs) from the distal proteome of the amyloplast and the proximal 'amylome' (the amyloplastic proteome), respectively. The term 'distal proteome' refers to SGAPs loosely tethered to the amyloplast, ones that can be rapidly proteolyzed, while proximal SGAPs are those found closer to the remnant amyloplast membrane fragments, perhaps embedded therein-ones that need isopropanol solvent to be removed from the mature organelle surface. These two rice starch-associated peptide samples were analyzed using nano-liquid chromatography-tandem mass spectrometry (Nano-HPLC-MS/MS). Known and novel proteins, as well as septum-like structure (SLS) proteins, in the mature rice SG were found. Data mining and gene ontology software were used to categorize these putative plastoskeletal components as a variety of structural elements, including actins, tubulins, tubulin-like proteins, and cementitious elements such as reticulata related-like (RER) proteins, tegument proteins, and lectins. Delineating the plastoskeletal proteome begins by understanding how each starch granule isolation procedure affects observed cytoplasmic and plastid proteins. The three methods described herein show how the technique used to isolate SGs differentially impacts the subsequent proteomic analysis and results obtained. It can thus be concluded that future investigations must make judicious decisions regarding the methodology used in extracting proteomic information from the compound starch granules being assessed, since different methods are shown to yield contrasting results herein. Data are available via ProteomeXchange with identifier PXD032314.

Fertilization time of slow-release fertilizer affects the physicochemical properties of starch from spring-sown waxy maize.

BACKGROUND: Slow-release fertilizer is widely used in cereal crop production because it is ecofriendly and laborsaving. Effects of different application stages (zero-, three-, and six-leaf stages, denoted as SN0, SN3, and SN6, respectively) of slow-release (N/P2 O5 /K2 O = 225/75/75 kg ha-1 ) fertilizer on physicochemical properties of starch from spring-sown waxy maize were investigated in 2018 and 2019. Application of traditional fertilizer (NCK, compound fertilizer; N/P2 O5 /K2 O = 75/75/75 kg ha-1 ) at sowing time and urea (N = 150 kg ha-1 ) at six-leaf stage was designated as the control. RESULTS: In comparison to the NCK, SN0 reduced grain starch content by 4.9%. Meanwhile, SN3 and SN6 did not affect this parameter. Nevertheless, all treatments, particularly SN6, increased average starch granule size. The slow-release fertilizer reduced proportion of chains with degree of polymerization (DP) > 24. Relative to NCK, SN6 increased starch crystallinity in both years, whereas SN0 and SN3 increased it in 2018 but reduced it in 2019. SN0 reduced peak, trough, and final viscosities, whereas SN3 and SN6 produced similar starch viscosities to those produced by NCK. No fertilizer mode affected gelatinization parameters, but SN6 produced a low retrogradation percentage. In comparison to data for 2018, starch produced in 2019 showed a small granule size, and a high proportion of short amylopectin chains. These properties endowed starch with high viscosity and low retrogradation percentage. CONCLUSION: In spring-sown waxy maize production, applying slow-release fertilizer at the six-leaf stage produced starch with high viscosity and low retrogradation tendency by enlarging granule size, increasing crystallinity, and reducing the proportion of long chains. © 2021 Society of Chemical Industry.

Starch Granules in Arabidopsis thaliana Mesophyll and Guard Cells Show Similar Morphology but Differences in Size and Number.

Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.

Starch Granule Size and Morphology of Arabidopsis thaliana Starch-Related Mutants Analyzed during Diurnal Rhythm and Development.

Transitory starch plays a central role in the life cycle of plants. Many aspects of this important metabolism remain unknown; however, starch granules provide insight into this persistent metabolic process. Therefore, monitoring alterations in starch granules with high temporal resolution provides one significant avenue to improve understanding. Here, a previously established method that combines LCSM and safranin-O staining for in vivo imaging of transitory starch granules in leaves of Arabidopsis thaliana was employed to demonstrate, for the first time, the alterations in starch granule size and morphology that occur both throughout the day and during leaf aging. Several starch-related mutants were included, which revealed differences among the generated granules. In ptst2 and sex1-8, the starch granules in old leaves were much larger than those in young leaves; however, the typical flattened discoid morphology was maintained. In ss4 and dpe2/phs1/ss4, the morphology of starch granules in young leaves was altered, with a more rounded shape observed. With leaf development, the starch granules became spherical exclusively in dpe2/phs1/ss4. Thus, the presented data provide new insights to contribute to the understanding of starch granule morphogenesis.

Water and heat stresses during grain formation affect the physicochemical properties of waxy maize starch.

BACKGROUND: Maize is frequently subjected to simultaneous water (drought or waterlogging) and heat (HS) stresses during grain formation in southern China. This work examined the effect of high temperature combined with drought (HD) or waterlogging (HW) during grain formation on the starch physicochemical properties of two waxy maize hybrids, namely Suyunnuo5 (SYN5) and Yunuo7 (YN7). RESULTS: Heat stress enlarged the starch granule size, and water stresses aggravated this effect. Heat stress reduced the ratio of small molecular weight fractions for both hybrids, and HD aggravated this reduction only in SYN5. Relative crystallinity in SYN5 was increased by stresses but in YN7 it was unaffected by HD, reduced by HS, and increased by HW. Fourier-transform infrared (FTIR) spectrometry results showed that the 1045/1022 cm-1 ratio in SYN5 was not influenced by HW but was increased by other stresses, and that in YN7 it was increased by all stresses, with the highest value induced by HW. Peak viscosity was decreased, whereas gelatinization temperatures and retrogradation percentage were increased by all of these stresses. These effects were exacerbated by combined heat and water stresses. The maximum decomposition rate was severely increased by HW. CONCLUSION: Drought or waterlogging at grain formation stage aggravated the detrimental effects of HS on the starch physicochemical properties of waxy maize. © 2020 Society of Chemical Industry.