Expression of the Granule-Bound Starch Synthase I (Waxy) Gene from Snapdragon Is Developmentally and Circadian Clock Regulated

The granule-bound starch synthase I (GBSSI or waxy) enzyme catalyzes one of the enzymatic steps of starch synthesis. This enzyme is responsible for the synthesis of amylose and is also involved in building the final structure of amylopectin. Little is known about expression of GBSSI genes in tissues other than storage organs, such as seeds, endosperm, and tuber. We have isolated a gene encoding the GBSSI from snapdragon (Antirrhinum majus). This gene is present as a single copy in the snapdragon genome. There is a precise spatial and developmental regulation of its expression in flowers. GBSSI expression was observed in all floral whorls at early developmental stages, but it was restricted to carpel before anthesis. These results give new insights into the role of starch in later reproductive events such as seed filling. In leaves the mRNA level of GBSSI is regulated by an endogenous circadian clock, indicating that the transition from day to night may be accompanied by abolition of expression of starch synthesis genes. This mechanism does not operate in sink tissues such as roots when grown in the dark.     

Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz)

In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.     

Molecular regulation of sink–source transition in rice leaf sheaths during the heading period

The upper leaf sheath of rice (Oryza sativa L.) serves as a temporary starch sink before heading, subsequently becoming a carbon source tissue to the growing panicle at the post-heading stage. The time of sink–source transition in upper leaf sheaths is highly correlated to the panicle exsertion. Here, we found that the expression profiles of starch synthesis genes such as ADP-glucose pyrophosphorylase large subunit 2, granule-bound starch synthase II, soluble starch synthase I, starch branching enzyme (SBE) I, SBEIII, and SBEIV were highly correlated with starch content changes during the heading period in the second leaf sheath below the flag leaf. In addition, the α-amylase2A and β-amylase were considered as major genes that were in charge of starch degradation at the post-heading period. Of the five sucrose transporter (OsSUT) genes, OsSUT1 and OsSUT4 appeared to play an important role in sucrose loading into the phloem of source leaf sheaths. Moreover, the microarray-based data implied that the dominant processes associated with functional leaf sheath transition from sink to source were carbohydrate metabolism and the translocation of the carbon and nitrogen sources and inorganic phosphate.     

Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis

The aim of this work was to investigate the effects on carbohydrate metabolism of a reduction in the capacity to degrade leaf starch in Arabidopsis. The major roles of leaf starch are to provide carbon for sucrose synthesis, respiration and, in developing leaves, for biosynthesis and growth. Wild-type plants were compared with plants of a starch-excess mutant line (sex4) deficient in a chloroplastic isoform of endoamylase. This mutant has a reduced capacity for starch degradation, leading to an imbalance between starch synthesis and degradation and the gradual accretion of starch as the leaves age. During the night the conversion of starch into sucrose in the mutant is impaired; the leaves of the mutant contained less sucrose than those of the wild type and there was less movement of ¹⁴C-label from starch to sucrose in radio-labelling experiments. Furthermore, the rate of assimilate export to the roots during the night was reduced in the mutant compared with the wild type. During the day however, photosynthetic partitioning was altered in the mutant, with less photosynthate partitioned into starch and more into sugars. Although the sucrose content of the leaves of the mutant was similar to the wild type during the day, the rate of export of sucrose to the roots was increased more than two-fold. The changes in carbohydrate metabolism in the mutant leaves during the day compensate partly for its reduced capacity to synthesize sucrose from starch during the night.     

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5 upstream sequence of the granule-bound starch synthase gene from potato and the -glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale(× Triticosecale Wittmack)

Triticale(× Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase,soluble starch synthases,granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development.There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale.Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development.Dead cells within the endosperm were detected at 6 d post anthesis(DPA),and evidence of DNA fragmentation was first observed at 21 DPA.The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development.Cell death occurred stochastically throughout the whole endosperm,meanwhile,the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling.These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

Multigene engineering of starch biosynthesis in maize endosperm increases the total starch content and the proportion of amylose

Maize (Zea mays spp. mays) is a staple crop for more than 900 million people. The seeds or kernels provide a rich source of calories because ~70 % of the weight is carbohydrate, mostly in the form of starch. The content and composition of starch are complex traits controlled by many genes, offering multiple potential targets for intervention. We used a multigene engineering approach combining the overexpression of Bt2, Sh2, Sh1 and GbssIIa (to enhance the activity of sucrose synthase, AGPase and granule-bound starch synthase) with the suppression of SbeI and SbeIIb by RNA interference (to reduce the activity of starch branching enzyme). Maize plants expressing all six genes plus the selectable marker showed a 2.8–7.7 % increase in the endosperm starch content and a 37.8–43.7 % increase in the proportion of amylose, which was significant compared to untransformed control plants. We also observed improvements in other agronomic traits, such as a 20.1–34.7 % increase in 100-grain weight, a 13.9–19.0 % increase in ear weight, and larger kernels with a better appearance, presumably reflecting the modified starch structure within the kernels. Our results confirm that multigene engineering applied to the starch biosynthesis pathway can not only modulate the quality and quantity of starch but can also improve starch-dependent agronomic traits.     

Systematic Analysis of Pericarp Starch Accumulation and Degradation during Wheat Caryopsis Development

Although wheat (Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development.     

Integrative Analysis of mRNA and miRNA Expression Profiles of the Tuberous Root Development at Seedling Stages in Turnips

The tuberous root of Brassica rapa L. (turnip) is an important modified organ for nutrition storage. A better understanding of the molecular mechanisms involved in the process of tuberous root development is of great value in both economic and biological context. In this study, we analyzed the expression profiles of both mRNAs and miRNAs in tuberous roots at an early stage before cortex splitting (ES), cortex splitting stage (CSS), and secondary root thickening stage (RTS) in turnip based on high-throughput sequencing technology. A large number of differentially expressed genes (DEGs) and several differentially expressed miRNAs (DEMs) were identified. Based on the DEG analysis, we propose that metabolism is the dominant pathway in both tuberous root initiation and secondary thickening process. The plant hormone signal transduction pathway may play a predominant role in regulating tuberous root initiation, while the starch and sucrose metabolism may be more important for the secondary thickening process. These hypotheses were partially supported by sequential DEM analyses. Of all DEMs, miR156a, miR157a, and miR172a exhibited relatively high expression levels, and were differentially expressed in both tuberous root initiation and the secondary thickening process with the expression profiles negatively correlated with those of their target genes. Our results suggest that these miRNAs play important roles in tuberous root development in turnips.     

Gibberellin disturbs the balance of endogenesis hormones and inhibits adventitious root development of Pseudostellaria heterophylla through regulating gene expression related to hormone synthesis

The adventitious roots of some plants will develop into tuberous roots which are widely used in many traditional Chinese medicines, including Pseudostellaria heterophylla. If adventitious root development is inhibited, the yield of Chinese medicinal materials will be reduced. Gibberellic acid is an important phytohormone that promotes plant growth and increases the resistance to drought, flood or disease. However, the effects of gibberellic acid on adventitious roots of Pseudostellaria heterophylla are not clear. Here, we reports GA3 suppressed adventitious root development of Pseudostellaria heterophylla by disturbing the balance of endogenesis hormones. By detecting the contents of various endogenous hormones, we found that the development of adventitious roots negatively correlated with the content of CA3 in tuberous roots. Exogenous GA3 treatment decreased the diameter of adventitious roots, but increased the length of adventitious roots of Pseudostellaria heterophylla. In contrast, blocking the biosynthesis of GA3 suppressed stem growth and promoted the xylem of tuberous roots development. Moreover, exogenous GA3 treatment resulted in imbalance of endogenesis hormones by regulating their synthesis-related genes expression in xylem of tuberous roots. These results suggest GA3 broke the established distribution of hormones by regulating synthesis, transport and biological activation of hormones to activate the apical meristem and suppress lateral meristem. Regulating GA3 signaling during adventitious roots development would be one of the possible ways to increase the yield of P. heterophylla.     

Enzymes involved in starch synthesis in the developing mung bean seed

The levels of free sugars, starch and enzymes involved in starch metabolism—sucrose synthetase, UDP and ADP glucose pyrophosphorylase, phosphorylase and starch synthetase—were assayed during seed development of three cultivars of mung bean (Vigna radiata). Free sugars and starch increased with increasing seed weight. Changes in levels of sucrose synthetase, UDP- and ADP-glucose pyrophosphorylases, and phosphorylase were paralleled by changes in starch accumulation. After the maximum activity levels of these enzymes had been reached, maximum activities of soluble starch synthetase and starch granule-bound starch synthetase occurred. There were high activities of sucrose synthetase and phosphorylase at maximum rates of starch accumulation. Thus, starch could be synthesized via the ADP glucose pathway in mung bean seeds. However, phosphorylase may account for the starch accumulation in the early stages of mung bean seed development.     

牛大力块根糖积累及其相关酶活性变化研究

为提高牛大力块根的产量与品质,该研究以不同发育时期(移栽6、12、18、24、30、36个月)的牛大力块根为材料,采用紫外分光光度法对糖类含量及其相关酶活性进行测定,研究它们在牛大力块根发育过程中的动态变化规律。结果表明:(1)牛大力块根的生长发育进程可初步划分为形成期(移栽6～12个月)、迅速膨大期(移栽12～24个月)与稳定膨大期(移栽24～36个月)三个阶段。淀粉与蔗糖分别是牛大力块根中主要的多糖与可溶性糖。在牛大力块根发育过程中,多糖类物质的含量逐渐增加,而可溶性糖含量逐渐减少,两者之间呈显著负相关,推测可溶性糖的分解代谢有利于促进多糖类物质的积累。(2)蔗糖的分解代谢是蔗糖合酶(SUS)、蔗糖磷酸合酶(SPS)、酸性转化酶(AI)与中性转化酶(NI)等多种相关酶协同作用的结果。SUS在牛大力块根发育过程中发挥着既催化蔗糖合成,又催化蔗糖分解的双重调节作用,SUS(合成)的活性不断上升,至移栽36个月达到峰值,极显著高于其他时期; SUS(分解)的活性从移栽6个月至24个月逐渐上升,但在块根稳定膨大期稍有下降; 其净活性为催化蔗糖分解,在移栽12个月达到最高。转化酶AI和NI的活性均在块根发育过程中逐渐上升,且AI活性高于NI活性,提示AI可能在蔗糖代谢分解过程中发挥更重要的作用。该研究结果可为今后深入研究牛大力多糖类成分积累和调控机制提供理论依据,并为提高牛大力药材的产量与品质提供技术指导。     

Response of sugar metabolism in the cotyledons and roots of Ricinus communis subjected to salt stress

• Ricinus communis is an important oilseed crop worldwide and is also considered one of the best potential plants for salt-affected soil improvement in northeast China. However, little is known about photosynthesis and carbohydrate metabolism in this plant, nor the distribution of carbohydrates in cotyledons and roots under salinity stress.
• In the present study, seedling growth, gas exchange parameters (P_N, E, g_s and C_i), carbohydrate (fructose, sucrose, glucose, soluble sugar and starch) metabolism and related enzymes and genes were measured in Ricinus plants.
• Under salt stress, P_N of cotyledons decreased significantly (P < 0.05), resulting in weak photosynthetic capacity. Furthermore, salt stress increased sucrose and glucose content in cotyledons, but decreased soluble sugar and starch content. However, sucrose increased and starch decreased in roots. This may be correlated with the increasing sugar metabolism under salinity, including notable changes in sugar-related enzyme activities (SPS, SuSy, α-amylase and β-amylase) and gene expression of RcINV, RcSUS, RcAmY, RcBAM and RcGBE1.
• The results suggest that salinity reduces photosynthesis of cotyledons, alters carbohydrate allocation between cotyledons and roots and also promotes starch utilization in cotyledons and starch biosynthesis in roots, leading to a functional imbalance between cotyledons and roots. Together, these findings provide insights into the crucial role of sugar metabolism in improving salt-tolerance of Ricinus during the early seedling growth stage.

     

Isolation and characterization of an alpha-amylase gene in cassava (Manihot esculenta).

The roots of cassava plants (Manihot esculenta Crantz) accumulate starch as their major form of carbohydrate reserve. Starch accumulation and properties are determined by a balance between starch biosynthesis and degradation processes. Alpha-amylases (EC 3.2.1.1) are alpha-1,4 endoglycolytic enzymes, responsible for the mobilization of stored carbohydrate reserves by initiating the degradation process. Alpha-amylase genes have been shown to be differentially expressed at various developmental stages and environmental conditions through the action of plant hormones such as abscisic acid (ABA) and gibberellic acid (GA). In this study, we isolated an alpha-amylase gene from cassava tuberous roots (designated as MEamy2, GenBank accession number DQ011041). The deduced product of MEamy2 is 407 amino acid residues in length, with a calculated molecular mass of 46.7 kDa and an isoelectric point of 8.66. Southern blot analysis showed that the MEamy2 is present as a single copy in cassava genome. It shares the highest homology with AMY8 from apple fruit. The predicted structural model of MEamy2 contains three domains, active sites and starch-binding domain that are common with other plant alpha-amylases. RT-PCR analysis showed that the MEamy2 gene expression was induced in cassava roots within 2 hours after treatment with GA, but not ABA.