Leaf starch degradation by ß-amylase ZmBAM8 influences drought tolerance in maize.

As a typical C4 plant and important crop worldwide, maize is susceptible to drought. In maize, transitory starch (TS) turnover occurs in the vascular bundle sheath of leaves, differing from that in Arabidopsis (a C3 plant). This process, particularly its role in drought tolerance and the key starch-hydrolyzing enzymes involved, is not fully understood. We discovered that the expression of the ß-amylase (BAM) gene ZmBAM8 is highly upregulated in the drought-tolerant inbred line Chang7-2t. Inspired by this finding, we systematically investigated TS degradation in maize lines, including Chang7-2t, Chang7-2, B104, and ZmBAM8 overexpression (OE) and knockout (KO) lines. We found that ZmBAM8 was significantly induced in the vascular bundle sheath by drought, osmotic stress, and abscisic acid. The stress-induced gene expression and chloroplast localization of ZmBAM8 align with the tissue and subcellular sites where TS turnover occurs. The recombinant ZmBAM8 was capable of effectively hydrolyzing leaf starch. Under drought conditions, the leaf starch in ZmBAM8-OE plants substantially decreased under light, while that in ZmBAM8-KO plants did not decrease. Compared with ZmBAM8-KO plants, ZmBAM8-OE plants exhibited increased drought tolerance. Our study provides insights into the significance of leaf starch degradation in C4 crops and contributes to the development of drought-resistant maize.

Characterization of a near isogenic barley line with high grain ß-amylase activity reveals a separation in the tight co-regulation of B-hordeins (Hor2) with endosperm-specific ß-amylase (Bmy1).

GSHO 2096 is a near isogenic barley line with extremely high grain ß-amylase activity, a desirable trait in the malting and brewing industry. High levels of grain ß-amylase activity are caused by a surge in endosperm-specific ß-amylase (Bmy1) gene expression during the early stages of grain development with high expression levels persisting throughout development. Origins of the high ß-amylase activity trait are perplexing considering GSHO 2096 is not supposed to have grain ß-amylase activity. GSHO 2096 is reported to be derived from a Bowman x Risø 1508 cross followed by recurrent backcrossing to Bowman (BC5). Risø 1508 carries a mutated form of the barley prolamin binding factor, which is responsible for Bmy1 expression during grain development. Thus, the pedigree of GSHO 2096 was explored to determine the potential origins of the high grain ß-amylase trait. Genotyping using the barley 50k iSelect SNP array revealed Bowman and GSHO 2096 were very similar (95.4 %) and provided evidence that both Risø 56 and 1508 are in the pedigree. Risø mutants 56 and 1508 both have perturbed hordein gene expression leading to a discernable pattern using SDS-PAGE. GSHO 2096 and Risø 56 have the same hordein pattern whereas Bowman and Risø 1508 have unique patterns. RNAseq revealed that Hor2 (B-hordein) gene expression was completely downregulated making it unique as the only known line with Bmy1 expression without Hor2 co-expression. Regardless of pedigree, GSHO 2096 remains an extremely valuable high ß-amylase activity line with potential utilization in breeding for malt quality.

Comparisons of the amylolytic enzymes and malt starch hydrolysates of two barley cultivars, Hokudai 1 (the first cultivar developed in Japan) and Kitanohoshi (currently used cultivar for beer production).

Starch degradation in malted barley produces yeast-fermentable sugars. In this study, we compared the amylolytic enzymes and composition of the malt starch hydrolysates of two barley cultivars, Hokudai 1 (the first cultivar established in Japan) and Kitanohoshi (the currently used cultivar for beer production). Hokudai 1 malt contained lower activity of amylolytic enzymes than Kitanohoshi malt, although these cultivars contained α-amylase AMY2 and ß-amylase Bmy1 as the predominant enzymes. Malt starch hydrolysate of Hokudai 1 contained more limit dextrin and less yeast-fermentable sugars than that of Kitanohoshi. In mixed malt saccharification, a high Hokudai 1 malt ratio increased the limit dextrin levels and decreased the maltotriose and maltose levels. Even though Kitanohoshi malt contained more amylolytic enzymes than Hokudai 1 malt, addition of Kitanohoshi extract containing the amylolytic enzymes did not enhance malt starch degradation of Hokudai 1. Hokudai 1 malt starch was less degradable than Kitanohoshi malt starch.

The effect of L-cysteine on starch and protein degradation during barley germination.

OBJECTIVES: In order to investigate the impact of L-cysteine (L-Cys) on starch and protein degradation during barley germination. The amylase activities, degradation of macromolecules during germination were determined in this study. METHODS: Barley was germinated in petri dish for 0 to 5 days with different levels of L-Cys (0 mM, 2.5 mM, 5 mM, 10 mM). RESULTS: L-Cys addition increased the total limit dextrinase (LD) activities and decreased the LD inhibitor activities during whole germination stage. The activities of α-amylase, ß-amylase and free LD were increased with the addition of 2.5, 5 mM L-Cys at germination days 1 to 4. Due to higher amylase in malt with the addition of L-Cys, the non-fermentable sugars were reduced and the glucose, maltotriose were improved. Furthermore, the protein degradation analysis showed that low molecular weight protein increased and middle molecular weight protein decreased obviously in wort from the malt germinated with L-Cys, demonstrating that the L-Cys promote the protein degradation. Lastly, the filtration performance of malt with the addition of L-Cys during malting was better than the control. CONCLUSION: In conclusion, L-Cys can promote the degradation of storage material (starch, protein) during barley germination, leading to a better green malt quality.

ACRE, a class of AP2/ERF transcription factors, activates the expression of sweet potato ß-amylase and sporamin genes through the sugar-responsible element CMSRE-1.

Sugars, synthesized by photosynthesis in source organs, are loaded and utilized as an energy source and carbon skeleton in sink organs, and also known to be important signal molecules regulating gene expression in higher plants. The expression of genes coding for sporamin and ß-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately induced by sugars. We previously reported on the identification of the carbohydrate metabolic signal-responsible element-1 (CMSRE-1) essential for the sugar-responsible expression of two genes. However, transcription factors that bind to this sequence have not been identified. In this study, we performed yeast one-hybrid screening using the sugar-responsible minimal promoter region of the ß-amylase gene as bait and a library composed only transcription factor cDNAs of Arabidopsis. Two clones, named Activator protein binding to CMSRE-1 (ACRE), encoding AP2/ERF transcription factors were isolated. ACRE showed transactivation activity of the sugar-responsible minimal promoter in a CMSRE-1-dependent manner in Arabidopsis protoplasts. Electric mobility shift assay (EMSA) using recombinant proteins and transient co-expression assay in Arabidopsis protoplasts revealed that ACRE could actually act to the CMSRE-1. Among the DEHYDRATION -RESPONSIVE ELEMENT BINDING FACTOR (DREB) subfamily, almost all homologs including ACRE, could act on the DRE, while only three ACREs could act to the CMSRE-1. Moreover, ACRE-homologs of Japanese morning glory also have the same property of DNA-binding preference and transactivation activity through the CMSRE-1. These findings suggested that ACRE plays an important role in the mechanism regulating the sugar-responsible gene expression through the CMSRE-1 conserved across plant species.

Overexpression of VaBAM3 from Vitis amurensis enhances seedling cold tolerance by promoting soluble sugar accumulation and reactive oxygen scavenging.

KEY MESSAGE: The VaBAM3 cloned from Vitis amurensis can enhance the cold tolerance of overexpressed plants, but VaBAM3 knock out by CRISPR/Cas9 system weakened grape callus cold tolerance. In grape production, extreme cold conditions can seriously threaten plant survival and fruit quality. Regulation of starch content by ß-amylase (BAM, EC: 3.2.1.2) contributes to cold tolerance in plants. In this study, we cloned the VaBAM3 gene from an extremely cold-tolerant grape, Vitis amurensis, and overexpressed it in tomato and Arabidopsis plants, as well as in grape callus for functional characterization. After exposure to cold stress, leaf wilting in the VaBAM3-overexpressing tomato plants was slightly less pronounced than that in wild-type tomato plants, and these plants were characterized by a significant accumulation of autophagosomes. Additionally, the VaBAM3-overexpressing Arabidopsis plants had a higher freezing tolerance than the wild-type counterparts. Under cold stress conditions, the activities of total amylase, BAM, peroxidase, superoxide dismutase, and catalase in VaBAM3-overexpressing plants were significantly higher than those in the corresponding wild-type plants. Furthermore, sucrose, glucose, and fructose contents in these lines were similarly significantly higher, whereas starch contents were reduced in comparison to the levels in the wild-type plants. Furthermore, we detected high CBF and COR gene expression levels in cold-stressed VaBAM3-overexpressing plants. Compared with those in VaBAM3-overexpressing grape callus, the aforementioned indicators tended to change in the opposite direction in grape callus with silenced VaBAM3. Collectively, our findings indicate that heterologous overexpression of VaBAM3 enhanced cold tolerance of plants by promoting the accumulation of soluble sugars and scavenging of excessive reactive oxygen species. These findings provide a theoretical basis for the cultivation of cold-resistant grape and support creation of germplasm resources for this purpose.

Glycogen ß-particles surface characterized by a combination of size exclusion chromatography and pyrene excimer fluorescence before and after ß-amylolysis.

Size exclusion chromatography (SEC) and pyrene excimer formation (PEF) experiments were conducted to characterize the local density profile inside a glycogen sample before (Glycogen) and after (Gly-ß-LD) treatment with ß-amylase. These experiments were conducted to assess whether the density at the periphery of the glycogen particles was very high to limit access to proteins involved in the metabolism of glycogen as predicted by the Tier model or low as suggested by the Gilbert model. SEC analysis indicated that the density inside the Glycogen and Gly-ß-LD samples remained constant with particle size and was not affected by ß-amylolysis. Analysis of the PEF experiments conducted on the Glycogen and Gly-ß-LD samples labeled with 1-pyrenebutyric acid showed that the particles have a dense interior and loose corona. The conclusions reached by the SEC and PEF experiments agree with the Gilbert model and have implications for the association of glycogen ß-particles into larger α-particles.

Pullulanase pretreatment of highly concentrated maltodextrin solution improves maltose yield during ß-amylase-catalyzed saccharification.

Increasing the substrate concentration can effectively reduce energy consumption and result in more economic benefits in the industrial production of maltose, but this process remarkably increases the viscosity, which has a negative effect on saccharification. To improve saccharification efficiency, pullulanase is usually employed. In the conventional process of maltose production, pullulanase is added at the same time with ß-amylase or later, but this process seems inefficient when the substrate concentration is high. Herein, a novel method was introduced to enhance the maltose yield under high substrate concentration. The results indicated that the pullulanase pretreatment of highly concentrated maltodextrin solution for 2 h greatly affects the final conversion rate of ß-amylase-catalyzed saccharification. The maltose yield reached 80.95 %, which is 11.8 % above the control value. Further examination confirmed that pullulanase pretreatment decreased the number of branch points of maltodextrin and resulted in a high content of oligosaccharides. These linear chains were suitable for ß-amylase-catalyzed saccharification to produce maltose. This research offers a new effective and green strategy for starch sugar production.

Genome-Wide Identification and Preliminary Functional Analysis of BAM (ß-Amylase) Gene Family in Upland Cotton.

The ß-amylase (BAM) gene family encodes important enzymes that catalyze the conversion of starch to maltose in various biological processes of plants and play essential roles in regulating the growth and development of multiple plants. So far, BAMs have been extensively studied in Arabidopsis thaliana (A. thaliana). However, the characteristics of the BAM gene family in the crucial economic crop, cotton, have not been reported. In this study, 27 GhBAM genes in the genome of Gossypium hirsutum L (G. hirsutum) were identified by genome-wide identification, and they were divided into three groups according to sequence similarity and phylogenetic relationship. The gene structure, chromosome distribution, and collinearity of all GhBAM genes identified in the genome of G. hirsutum were analyzed. Further sequence alignment of the core domain of glucosyl hydrolase showed that all GhBAM family genes had the glycosyl hydrolase family 14 domain. We identified the BAM gene GhBAM7 and preliminarily investigated its function by transcriptional sequencing analysis, qRT-PCR, and subcellular localization. These results suggested that the GhBAM7 gene may influence fiber strength during fiber development. This systematic analysis provides new insight into the transcriptional characteristics of BAM genes in G. hirsutum. It may lay the foundation for further study of the function of these genes.

The LIKE SEX FOUR 1-malate dehydrogenase complex functions as a scaffold to recruit ß-amylase to promote starch degradation.

In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including ß-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit ß-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex. The starch hydrolysis activity of BAM1 drastically increased in the presence of LSF1-MDH in vitro. We determined the structure of the BAM1-LSF1-MDH complex by a combination of cryo-electron microscopy, crosslinking mass spectrometry, and molecular docking. The starch-binding domain of the dual-specificity phosphatase and carbohydrate-binding module of LSF1 was docked in proximity to BAM1, thus facilitating BAM1 access to and hydrolysis of the polyglucans of starch, thus revealing the molecular mechanism by which the LSF1-MDH complex improves the starch degradation activity of BAM1. Moreover, LSF1 is phosphatase inactive, and the enzymatic activity of MDH was dispensable for starch degradation, suggesting nonenzymatic scaffold functions for LSF1-MDH in starch degradation. These findings provide important insights into the precise regulation of starch degradation.

Genome-Wide Investigation of BAM Gene Family in Annona atemoya: Evolution and Expression Network Profiles during Fruit Ripening.

ß-amylase proteins (BAM) are important to many aspects of physiological process such as starch degradation. However, little information was available about the BAM genes in Annona atemoya, an important tropical fruit. Seven BAM genes containing the conservative domain of glycoside hydrolase family 14 (PF01373) were identified with Annona atemoya genome, and these BAM genes can be divided into four groups. Subcellular localization analysis revealed that AaBAM3 and AaBAM9 were located in the chloroplast, and AaBAM1.2 was located in the cell membrane and the chloroplast. The AaBAMs belonging to Subfamily I contribute to starch degradation have the higher expression than those belonging to Subfamily II. The analysis of the expression showed that AaBAM3 may function in the whole fruit ripening process, and AaBAM1.2 may be important to starch degradation in other organs. Temperature and ethylene affect the expression of major AaBAM genes in Subfamily I during fruit ripening. These expressions and subcellular localization results indicating ß-amylase play an important role in starch degradation.

Humulus lupulus and microbes: Exploring biotic causes for hop creep.

BACKGROUND: Hop creep continues to present an unresolved issue for the brewing industry, specifically stemming from those hops added to beer during fermentation. Hops have been found to contain four dextrin-degrading enzymes: alpha amylase, beta amylase, limit dextrinase, and an amyloglucosidase. One recent hypothesis predicts that these dextrin-degrading enzymes could originate from microbes rather than the hop plant itself. SCOPE AND APPROACH: This review begins by describing how hops are processed and used in the brewing industry. It will then discuss hop creep's origins with a new beer style, antimicrobial factors from hops and resistance mechanisms that bacteria use to counter them, and finally microbial communities that inhabit hops, focusing on whether they can produce the starch degrading enzymes which drive hop creep. After initial identification, microbes with possible links to hop creep were then run through several databases to search the genomes (if available) and for those specific enzymes. KEY FINDINGS AND CONCLUSIONS: Several bacteria and fungi contain alpha amylase as well as unspecified glycosyl hydrolases, but only one contains beta amylase. Finally, this paper closes with a short summary of how abundant these organisms typically are in other flowers.

Immunoglobulin E-specific allergens against leaf in serum of dogs with clinical features of grass leaf allergy.

BACKGROUND: Grass leaf has been suspected of causing immunoglobulin (Ig)E-mediated immediate hypersensitivity reactions in humans and dogs. However, most studies in this area are case-control studies without in vitro data showing the involvement of IgE in the reaction. Laboratory studies have demonstrated the reactivity to a 50-55 kDa protein with clinical signs immediately after contact with grass leaf material. The clinical findings of dogs with atopic-like dermatitis immediately after contact with grass leaf material suggest the involvement of grass leaves as the allergen source. OBJECTIVES: This study was designed to test the IgE-reactivity of grass leaf proteins in dogs with clinical signs and positive scratch test results against grass leaf material. MATERIALS AND METHODS: The serum of 41 patients with a history of allergy and suspected to grass leaf material was immunoblotted against grass leaf extracts from five suspected grass species. The IgE-positive blots were separated with 2D gel electrophoresis and analysed with mass spectrometry (MS). Commercially supplied proteins were used to validate immunoblot activity. RESULTS: The serum of 25 dogs diagnosed with grass dermatitis had positive IgE-specific immunoblot against one or more grass leaf extracts. The MS data indicated a reactive band at 55 kDa to be beta-amylase or RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit (RbLS). All tested dog sera showed IgE-reactivity with beta-amylase and some with RbLS. CONCLUSIONS AND CLINICAL RELEVANCE: Canines with clinical signs of grass-related dermatitis had IgE-reactivity against grass leaf proteins. Serum IgE-reactivity to beta-amylase and RuBisCO large subunit may indicate that these proteins act as allergens, possibly causing pruritus and skin lesions.

The relationship between ß-amylase and the degradation of starch temporarily stored in rice leaf blades.

Starch is stored temporarily in the leaves during the day but degraded during the night. In this study, we investigated the relationship between diurnal changes in starch content in rice leaf blades and the mRNA levels of ß-amylase genes. In addition to the known plastid-type ß-amylases OsBAM2 and OsBAM3, OsBAM4, and OsBAM5 were also identified as plastid targeted proteins. In the leaf blades, starch contents, which reached its maximum at the end of day, showed two periods of marked decrease: from 18:00 to 21:00 and from 24:00 to 6:00. The expression of OsBAM2, OsBAM3, OsBAM4, and OsBAM5 was maintained at a low level from 18:00 to 21:00 but increased strongly after midnight. Furthermore, ß-amylase activity gradually increased after 21:00, reaching a maximum during the early morning. These results suggest that in rice leaf blades, ß-amylase plays an important role in starch degradation by being highly active from midnight to dawn.

Genome-wide characterization and expression analysis of α-amylase and ß-amylase genes underlying drought tolerance in cassava.

BACKGROUND: Starch hydrolysates are energy sources for plant growth and development, regulate osmotic pressure and transmit signals in response to both biological and abiotic stresses. The α-amylase (AMY) and the ß-amylase (BAM) are important enzymes that catalyze the hydrolysis of plant starch. Cassava (Manihot esculenta Crantz) is treated as one of the most drought-tolerant crops. However, the mechanisms of how AMY and BAM respond to drought in cassava are still unknown. RESULTS: Six MeAMY genes and ten MeBAM genes were identified and characterized in the cassava genome. Both MeAMY and MeBAM gene families contain four genes with alternative splicing. Tandem and fragment replications play important roles in the amplification of MeAMY and MeBAM genes. Both MeBAM5 and MeBAM10 have a BZR1/BES1 domain at the N-terminus, which may have transcription factor functions. The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress. MeAMY1, MeAMY2, MeAMY5, and MeBAM3 are proven as critical genes in response to drought stress according to their expression patterns under drought. The starch content, soluble sugar content, and amylase activity were significantly altered in cassava under different levels of drought stress. CONCLUSIONS: These results provide fundamental knowledge for not only further exploring the starch metabolism functions of cassava under drought stress but also offering new perspectives for understanding the mechanism of how cassava survives and develops under drought.

Study on the Interaction Mechanism of Methoxy Polyethylene Glycol Maleimide with Sweet Potato ß-Amylase.

In this study, sweet potato ß-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified ß-amylase and the interaction mechanism between SPA and Mal-mPEG5000 was investigated. the changes in the functional groups of different amide bands and modifications in the secondary structure of enzyme protein were analyzed using infrared spectroscopy and circular dichroism spectroscopy. The addition of Mal-mPEG5000 transformed the random curl in the SPA secondary structure into a helix structure, forming a folded structure. The Mal-mPEG5000 improved the thermal stability of SPA and protected the structure of the protein from breaking by the surrounding. The thermodynamic analysis further implied that the intermolecular forces between SPA and Mal-mPEG5000 were hydrophobic interactions and hydrogen bonds due to the positive values of ΔHθ and ΔSθ. Furthermore, the calorie titration data showed that the binding stoichiometry for the complexation of Mal-mPEG5000 to SPA was 1.26, and the binding constant was 1.256 × 107 mol/L. The binding reaction resulted from negative enthalpy, indicating that the interaction of SPA and Mal-mPEG5000 was induced by the van der Waals force and hydrogen bonding. The UV results showed the formation of non-luminescent material during the interaction, the Fluorescence results confirmed that the mechanism between SPA and Mal-mPEG5000 was static quenching. According to the fluorescence quenching measurement, the binding constant (KA) values were 4.65 × 104 L·mol-1 (298K), 5.56 × 104 L·mol-1 (308K), and 6.91 × 104 L·mol-1 (318K), respectively.

Mepiquat chloride application combined with high plant population density promotes carbon remobilization in the roots of upland cotton.

Carbon reserves in cotton roots can be remobilized to support reproductive growth, thus potentially affecting cotton yield. However, the regulation of carbon remobilization in cotton roots and its relationship with cotton yield are still poorly understood. Plant population density (PPD) and mepiquat chloride (MC) have been hypothesized to affect the dynamics of nonstructural carbohydrate content and the resulting carbon remobilization in cotton roots through the regulation of carbohydrate metabolism enzyme activities. A mid-maturation cotton line 4003-6 was field-grown in 2019 and 2020. Three different levels of PPD (D1: 2.25 plants m-2, D2: 4.5 plants m-2, and D3: 6.75 plants m-2) and two levels of MC dosage (M0: 0 g hm-2, M1: 82.5 g hm-2) were combined to create six populations differing in terms of the source-sink relationship. The changes in the hexose, sucrose, and starch contents and the key carbon metabolic enzyme activities in cotton roots were examined during peak squaring (PS) to late boll opening (LB). The combination of the PPD of 6.75 plants m-2 and MC application (M1D3) exhibited the greatest cotton yield and reproductive biomass-to-leaf area ratio from peak flowering (PF) onwards. M1D3 presented the greatest total nonstructural carbohydrate (TNC) remobilization amount of 2.96 and 3.80 g m-2, the highest efficiency of 39.11% and 48.39%, and the largest gross contribution to seed cotton yield of 0.66% and 0.79% in 2019 and 2020, respectively. The three parameters were positively correlated with the seed cotton yield except for the remobilization rate in 2019. Unlike the other treatments, the greater carbohydrate content per unit ground area in M1D3 prior to the PF stage was attributed to the higher sucrose phosphate synthase (SPS) and ADP-glucose pyrophosphorylase (AGPase) activities during the PS to first flowering (FF) stages. Conversely, the greater α-amylase and ß-amylase activities in M1D3 at the PF stage accounted for the lower starch content at the EB stage, and the smaller vacuolar invertase (VIN) and cell wall invertase (CWIN) activities at the EB stage could be responsible for the lower hexose concentration at that time. The TNC remobilization amount had a positive association with the AGPase and SPS activities at the FF stage and with ß-amylase activity at the PF stage in cotton tap roots in 2019 and 2020. This study provides a cotton yield-improving alternative through the promotion of carbon remobilization in roots using certain agronomic practices.

Transcription factors ABF4 and ABR1 synergistically regulate amylase-mediated starch catabolism in drought tolerance.

ß-Amylase (BAM)-mediated starch degradation is a main source of soluble sugars that help plants adapt to environmental stresses. Here, we demonstrate that dehydration-induced expression of PtrBAM3 in trifoliate orange (Poncirus trifoliata (L.) Raf.) functions positively in drought tolerance via modulation of starch catabolism. Two transcription factors, PtrABF4 (P. trifoliata abscisic acid-responsive element-binding factor 4) and PtrABR1 (P. trifoliata ABA repressor 1), were identified as upstream transcriptional activators of PtrBAM3 through yeast one-hybrid library screening and protein-DNA interaction assays. Both PtrABF4 and PtrABR1 played a positive role in plant drought tolerance by modulating soluble sugar accumulation derived from BAM3-mediated starch decomposition. In addition, PtrABF4 could directly regulate PtrABR1 expression by binding to its promoter, leading to a regulatory cascade to reinforce the activation of PtrBAM3. Moreover, PtrABF4 physically interacted with PtrABR1 to form a protein complex that further promoted the transcriptional regulation of PtrBAM3. Taken together, our finding reveals that a transcriptional cascade composed of ABF4 and ABR1 works synergistically to upregulate BAM3 expression and starch catabolism in response to drought condition. The results shed light on the understanding of the regulatory molecular mechanisms underlying BAM-mediated soluble sugar accumulation for rendering drought tolerance in plants.

VaBAM1 weakens cold tolerance by interacting with the negative regulator VaSR1 to suppress ß-amylase expression.

Cold stress is a key climatic factor that limits grape productivity and quality. Although ß-amylase (BAM) is known to play an important role as a mediator of starch degradation under conditions of cold stress, the mechanism by which BAM regulates cold tolerance in grape remains unclear. Here, we identified VaBAM1 from Vitis amurensis and characterized its interactive regulating mechanism under cold stress in Arabidopsis thaliana and grape. VaBAM1-overexpressing A. thaliana plants (OEs) exhibited high freezing tolerance. Soluble sugar content and amylase activity were increased in OEs and VaBAM1-overexpressing grape calli (VaBAM1-OEs) under cold stress; however, they were decreased in grape calli in which VaBAM1 was edited using CRISPR/Cas9. The results of yeast two-hybrid, bimolecular fluorescence complementation, and pull-down experiments showed that serine/arginine-rich splicing factor 1 (VaSR1) interacted with VaBAM1. Furthermore, the expression of VaSR1 was opposite that of VaBAM1 in phloem tissue of Vitis amurensis during winter dormancy. In VaSR1-overexpressing grape calli (VaSR1-OEs), BAM activity and the expression levels of C-repeat binding transcription factor and cold response genes were all significantly lower than that in untransformed calli subjected to cold stress. Moreover, VvBAM1 was downregulated in VaSR1-OEs under cold stress. Overall, we identified that VaSR1 interacts with VaBAM1, negatively regulating BAM activity and resulting in decreased plant cold tolerance.

Co-Overexpression of Two Key Source Genes, OsBMY4 and OsISA3, Improves Multiple Key Traits of Rice Seeds.

Optimized source-sink interactions are determinants of both rice yield and quality. However, most source genes have not been well studied in rice, a major grain crop. In this study, OsBMY4 and OsISA3, the key ß-amylase and debranching enzymes that control transient starch degradation in rice leaves, were co-overexpressed in rice in order to accelerate starch degradation efficiency and increase the sugar supply for sink organs. Systematic analyses of the transgenic rice indicated that co-overexpression of OsBMY4 and OsISA3 not only promoted rice yield and quality, but also improved seed germination and stress tolerance. Moreover, since the OsBMY4 gene has not been characterized, we generated osbmy4 mutants using CRIPSR/Cas9 gene editing, which helped to reveal the roles of ß-amylase in rice yield and quality. This study demonstrated that specific modulation of the expression of some key source genes improves the source-sink balance and leads to improvements in multiple key traits of rice seeds.