Research progress of plant sucrase

Si Lizhen (1) is stored as an adult talent (2)

(Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing10010/)

In most higher plants, sucrose is the main transportation form of carbohydrate assimilation products from source to sink. In the library, sucrase can hydrolyze sucrose into glucose and fructose to meet the demand of plant growth and development for carbon source and energy. This paper summarizes some research progress of sucrase in recent years, including its classification, basic properties, gene structure,

Regulation of enzyme activity and function.

Keywords plant, sucrase, activity regulation, function

It is called extracellular sucrase. It is most necessary for different sucrase reactions.

Introduction to 0

Plants fix C O 2 in leaves (source tissues) through photosynthesis.

It is defined as carbohydrates and then transported to non-photosynthetic tissues (sink tissues). Most plants transport carbohydrate assimilation products from the source to the sink in the form of non-reducing disaccharides such as sucrose. In sink tissue, sucrose is decomposed into hexose, which provides carbon source and energy for plant growth and development. Sucrose decomposition is mainly completed by sucrose synthase (EC2). 4. 1. 13) or sucrase (E C3. 2. 1.26). Sucrose synthase is a glycosyltransferase that converts sucrose into uridine diphosphate glucose and fructose in the presence of UDP. Sucrase is a hydrolase that hydrolyzes sucrose into glucose and fructose. Sucrase has many isoenzymes, which are located in different subcellular locations and have different biochemical characteristics [1, 2]. Although their functional specificity is not clear, it is known that sucrase is mainly involved in regulating the different utilization modes of sucrose in plants. Because sugar is not only used as energy in plants, but also one of the important regulatory substances of gene expression, sucrase is also indirectly involved in the regulation of cell differentiation and plant development. In view of this, the study of sucrase is of great significance both in theory and in practice, and has attracted much attention. This paper introduces the research progress in recent years.

The suitable pH value is also different, so sucrase can be divided into acidic sucrase and neutral/alkaline sucrase. Liquid cell sucrase and cell wall sucrase have the highest catalytic efficiency at pH 4. Five to five. 0, so it is also called acid sucrase. The optimum pH value of cytoplasmic sucrase is neutral or slightly alkaline, so it is called neutral/alkaline sucrase. Sucrase can be divided into soluble sucrase (including liquid cell sucrase and cytoplasmic sucrase) and insoluble sucrase (cell wall sucrase) according to its solubility.

2 Basic properties of sucrase

Most plants contain at least two kinds of liquid cell sucrase, both of which exist in the form of soluble protein. Cell wall sucrase is bound to the cell wall in the form of ionic bonds, and there are many isozymes. Liquid cell type and cell wall type sucrase have the highest efficiency at pH4. Five to five. 0, and attacks sucrose from fructose residue, so this acidic sucrase is called β-fructofuranosidase. Because of this, acid sucrase can also catalyze the hydrolysis of other β -fructose containing polysaccharides, such as stachyose and raffinose. At present, acid sucrase has been isolated from many plants, and the molecular weight of mature peptides is mostly between 55 and 70 KD. Denatured SDS gel electrophoresis showed that 70kD liquid cell sucrase could be hydrolyzed into 30kD N-terminal and 38kD C-terminal. They have a Km value when the sucrose concentration is very low, and the enzyme activity is inhibited by heavy metal ions such as Hg2+ and Ag 2+, indicating that sulfhydryl groups exist in the catalytic center. Acid sucrase is also inhibited by its reaction products, glucose is a non-competitive inhibitor and fructose is a competitive inhibitor [3].

In addition, there are at least two kinds of cytoplasmic sucrase in plants, and the optimum pH for hydrolyzing sucrose is neutral or alkaline. Neutral or biased

Classification of 1 sucrase

According to its subcellular position in plants, sucrase can be divided into liquid cell sucrase, cytoplasmic sucrase and cell wall sucrase. The first two are also called intracellular sucrase, and cell wall sucrase is also called intracellular sucrase.

① Female, born in 1974, doctoral student; Research direction: molecular genetics.

② Contact person.

-03-29 (date of receipt: 2002)

Hi-tech Newsletter August 2002

The characteristics of alkaline sucrase are not clear, but compared with acidic sucrase, these enzymes have sucrose specificity. Alkaline sucrase mostly exists in the form of homotetramer, with a molecular weight of about 54 ~ 65 KD, and its Km (sucrose) is10 mmol/L. The enzyme activity is inhibited by the reaction products glucose and fructose, but not by heavy metal ions [4]. Because it is difficult to isolate neutral sucrase, there is little research on them.

Glucosidase analysis showed that their expression was organ-specific, Lin6 was sink-tissue-specific, while Lin5 and Lin7 were stamen-specific and pistil-specific, indicating that these two genes were related to carbon in flower organs.

Supply water assimilation products [13]. In tomato, 1 liquid cell sucrase gene and four cell wall sucrase genes have also been isolated, and their expressions are organ-specific [14]. This shows that acid sucrase in plants is encoded by a small gene family, and these genes are expressed independently in a specific time and space during plant development.

Besides organ and developmental specific expression, sugar composition and concentration

Acid sucrase has been isolated from tomato, Arabidopsis thaliana, maize and Hu.

Radish [7] was cloned, and its gene structure was basically the same, containing 6 ~ 8 exons [2]. Except the cell wall sucrase of carrot [7], all genes contain a tiny exon, which only encodes a tripeptide (DPN) in the center of NDPNG and a conservative element. In potato, this smallest exon has a special cross feature [8]. When invertase gene was expressed normally, no post-transcriptional processing errors were found. However, under cold stress, the RNA shearing process was destroyed and the exons of the transcripts were deleted. It is not clear whether this shear anomaly has physiological and biochemical effects. The amino acid sequence analysis of acid sucrase showed that there was a signal peptide and a propeptide at the N- terminal. The function of the propeptide is not clear, and it is speculated that it may be related to protein folding and protein localization [9], or to the control of enzyme activity [10].

There is little research on neutral/alkaline sucrase. The neutral sucrase cloned from carrot has no signal peptide at the N-terminal, is rich in cysteine, and has low homology with acid sucrase [1 1].

[5]

[2]

[6]

3 gene structure of sucrase

Degree can also significantly affect the expression of these acidic sucrase. no

Different sucrase in the same plant, the same plant has different reactions to sugar. At present, two genes with different reactions have been identified. The first type increases the expression with the increase of carbohydrates, and the other type negatively regulates them, reducing the content of sucrose and increasing the expression, and has similar reactions to glucose, sucrose and other metabolic sugars. Interestingly, the expression of soluble acid sucrase gene in American quinoa cells was not affected by sugar, but the expression of cell wall sucrase gene increased with the increase of sugar content. In carrots, sugar has no effect on acid sucrase. It is not clear why different acid sucrase genes in different plants have different responses to sucrose. One possibility is that in plants, such as carrots, plants store a lot of sugar, so the sugar content is unfavorable as a regulator of acid invertase, and the regulatory function of sugar may not be developed or lost in evolution. In these plants, short-term physiological changes only lead to slight changes in sugar content, which is not enough to effectively regulate the expression of sucrase gene.

In many plants, plant growth regulators such as auxin [17], gibberellin [18] or cytokinin can also improve the activity of acid invertase. At present, it is not clear whether plant hormones directly regulate the expression of invertase, or whether hormones lead to cell proliferation and produce new bank tissues. In the tissue culture cells of Atriplex americana, due to the influence of cytokinin, the transcription of cell wall sucrase and glucose carrier increased at the same time [19], which is probably due to the increase of carbon source supply caused by hormone stimulation of cell proliferation. Damage can also regulate the expression of acid sucrase gene. For example, the activity of soluble acid sucrase increased significantly in mature potato tuber slices [20], reached a peak at 18 hours, and then decreased. When the root cap of carrot was mechanically damaged, the expression of sucrase in its cell wall also changed, and mR NA reached the highest value at 12 hour after injury, accompanied by the increase of enzyme activity. The effect of injury on its expression is not systematic, and the change of activity is limited to the injured site.

There are also many reports on the relationship between the increase of acid sucrase activity and infection of different diseases [2 1]. In the root cap of carrot, it is connected with virus.

[2 1]

[ 19]

[ 16]

[ 15]

Regulation of sucrase

Because the activity of neutral/alkaline sucrase is easy to be lost in the extraction process, there is little research on them. There is a lot of information about the regulation of acid sucrase.

In Arabidopsis thaliana, acid sucrase is encoded by at least four genes: at be-tafuc 1, fruc2, fruc3 and fruc4, including cell wall sucrase gene and liquid cell sucrase gene. The analysis of their expression patterns shows that their expression has organ or developmental specificity [12]. The cell wall type of βfr UC 1 gene has high expression activity in mature leaves, but it is not expressed in cotyledons and flower organs. Specific expression of atβfr uc2 gene in flower organs. The expression pattern of type A fruc3 in liquid cells is completely different from that of the first two cell wall sucrase. Northern and RT -PCR results showed that the gene was highly expressed in cotyledons, but low expressed in mature leaves, roots and flower organs. In βfr, uc 4 is expressed in young leaves, but not in mature leaves. Potato extracellular sugarcane [1]

Research progress of plant sucrase

After touching 1 hour, the transcription level of sucrase reached the highest, and then decreased rapidly. Like injury, the influence of disease infiltration on its expression is not systematic, and the change of activity is limited to infiltration.

Location.

This change not only shows that sucrose needs to be converted into monosaccharide during cell elongation, but also shows that sucrose needs to be used as metabolic substrate during cell proliferation. It can be seen that vacuole sucrase plays an important role in plant growth by regulating sucrose.

Sucrase also plays a role in maintaining the normal function of cells under unfavorable conditions. Such as non-seasonal rainfall, high temperature, delayed harvest, etc. Can lead to the retransfer of sucrose to adapt to external changes [28]. In transgenic tobacco, the expression of yeast sucrase improved the salt tolerance of plants, and the photosynthesis of plants did not change under salt stress, while the photosynthesis of wild type was inhibited under the same conditions. Further analysis showed that the osmotic pressure of transgenic plants was significantly higher than that of the control, indicating that sucrase could improve the salt tolerance of plants by regulating the osmotic pressure of plants.

Like intracellular sucrase, extracellular sucrase also plays an important role in regulating the distribution of assimilates and resisting adversity [30]. The role of extracellular sucrase in signal transduction will help to clarify its role in source-sink regulation. In transgenic carrots, extracellular sucrase and liquid cell acid sucrase play an important role in plant growth and development [3 1], especially in the early development of plants. 5.2 Adjustment of source-sink relationship

The regulating function of acid sucrase in regulating source-sink relationship has been confirmed in model plants such as tobacco, Arabidopsis, potato and tomato by molecular manipulation technology. Overexpression of yeast sucrase in different parts of subcellular proves that it plays an important role in the long-distance transportation of photosynthetic products [32, 33]. No matter whether yeast sucrase is expressed in liquid cells or cytoplasm of tobacco, transgenic plants show abnormal growth, carbohydrate accumulation in source leaves, delayed root formation and severe dwarfing, and starch and soluble sugar accumulate in leaves, which indicates that the sugar transport balance of transgenic plants is destroyed. When sucrase is expressed in cytoplasm, starch and soluble sugar accumulate in source leaves and sink leaves, and leaves curl, indicating that cell division and cell growth are relatively fast at the top of leaves. In the source leaves of potato, yeast sucrase was over-expressed, and transgenic plants showed atrophy of leaves, stunted growth and reduced tuber number. At the cellular level, the contents of hexose, starch and amino acids in source leaves increased, especially proline, which was 40 times higher than that of wild type. The photosynthetic rate of transgenic plants decreased, osmotic pressure increased, and the protein content of 1, 5- rubp carboxylase (Rubisco) remained unchanged, but the proportion and catalytic efficiency decreased in the activated state [34]. The same phenotype was observed in transgenic tobacco, Arabidopsis thaliana and tomato, although the degree of inhibition was different in different plants [2]. However, in the cytoplasm of potato tuber,

[33]

[29]

5 the function of sucrase

5. 1 Regulation of plant growth

Sucrose has many important functions in plants (nutrition, osmotic adjustment, signal molecules), and so does sucrase.

In most cases, sucrase hydrolyzes sucrose into hexose for respiratory consumption, or as a carbon source and energy to synthesize many other compounds. Sucrase can participate in the long-distance transportation of sucrose in phloem [22]. In addition, sucrose can be decomposed into glucose and fructose by sucrase, which can greatly improve the osmotic pressure of cells, indicating that sucrose may play a role in cell elongation and plant growth [23].

In most studies, the function of sucrase is inferred by measuring the activity of sucrase in physiological process. For example, in the fast-growing carrot roots or bean stalks, the activity of sucrase is very high, but the sucrose content in these tissues is very low or can be rapidly reduced, so the function of acid sucrase in these tissues is to hydrolyze sucrose to meet the high demand of plants for reducing sugar [4]. The expression of yeast sucrase in pea cotyledons leads to the decrease of sucrose content and the accumulation of hexose. Cells of transgenic plants contain large vacuoles, which can last for a long time. However, in the wild type, large vacuoles are often replaced by small protein. When the plant becomes mature or dehydrated, the transgenic plant appears plasma-wall separation and vesicles are formed in the intima system [24]. The growth of plant tissues not only needs hexose as carbon source and energy source, but also needs a driving force of cell elongation, that is, the osmotic pressure of cells is relatively stable at ordinary times, but increases when the cell wall elongates. By analyzing the reaction between the upper and lower parts of occipital lobe under the action of gravity, an indirect evidence that soluble acid sucrase plays a role in osmotic adjustment was obtained. In the lower part, invertase activity increased by 3 times, which proved that a potential driving force of plant growth was osmotic pressure gradient.

In the developing sunflower hypocotyl, it was found that there was a strong positive correlation between cell elongation rate and acid sucrase activity [4]. The research by Woodson and Wang (1987) shows that the increase of soluble acid sucrase activity and reducing sugar level in blooming carnation flowers is a very important factor to regulate the growth of carnation petals, and these reducing sugars mainly come from sucrose input and subsequent hydrolysis [25].

In sugarcane, the activity of liquid cell acid sucrase is very high in immature and vigorous stem tissues, and its activity decreases with the increase of stem maturity [26, 27]. Liquid cell acid sucrase

[23]

Hi-tech Newsletter August 2002

Heterologous expression of invertase in yeast leads to smaller tubers and lower yield, but the number of tubers increases. If this gene is expressed in tuber cells, the result is just the opposite, that is, the tuber becomes larger, the number decreases, but the yield increases. The expression of acid sucrase in tomato cells was reduced by antisense technology. Although the plant growth showed no abnormality, the fruit became smaller [35]. The above results show that the size of sink can be adjusted by changing sucrose metabolism.

Yeast sucrase gene was introduced into sugarcane callus under the control of ubiquitin promoter.

[38][36, 37]

The specific expression of yeast sucrase in cytoplasm and cells of potato tuber leads to the decrease of sucrose content and the increase of glucose content. The increase of sucrase activity in tuber cytoplasm accelerated the speed of glycolysis.

Degree, leading to the accumulation of phosphorylated intermediates, but these phosphorylated intermediates have not been used to synthesize starch. In addition, the intercellular expression of sucrase does not increase the content of hexose phosphate [37].

6 problems and prospects

At present, the research of acid sucrase has made great progress. Not only the sucrase was purified, but also the activity determination method was established. In addition, their cDNA has been cloned from many plants and expressed and identified in transgenic plants. However, there are still many problems to be solved for sucrase in plants, such as: 1) Why sucrase in different subcellulars has different characteristics and how to coordinate it? 2) What is the function of neutral/alkaline sucrase? 3) Whether there are other metabolites or substances that regulate the expression of sucrase, and so on. This requires the organic combination of physiology, biochemistry and molecular biotechnology. The final solution to these problems will help to better manipulate the metabolism and distribution of carbohydrates in plants.

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In the cytoplasm and cytoplasm of these cells.

Sucrase activity increased, but sucrose accumulation decreased. Pass the counter

Just expression, reducing the activity of acid sucrase in transgenic tomatoes, can change the ratio of sucrose to hexose [35, 39]. Although there is no difference in phenotype between transgenic plants and wild type, the fruit is reduced by 30% compared with the control, which indicates that the gene plays an important role in the development of the library. 5.3 Regulation of Sucrose Distribution in Repository

A controversial issue is whether sucrase is involved in sucrose metabolism of sink organs such as seeds, tubers and roots. Sucrose synthase is the main enzyme that degrades sucrose during the development of Lyme pea seeds and potato tubers, and the amount of sucrose degradation by sucrase is very low [40]. This conclusion is also consistent with the research results of wheat endosperm development. Sucrose synthase is active in the whole endosperm.

[42]

The activity of sucrase during development is higher than that of sucrase. Weber et al. (1995) came to the opposite conclusion when studying the emission and distribution mechanism of photosynthetic products during seed development. During pre-storage, they found that the high level of hexose content in cotyledons and endosperm was positively correlated with the high cell wall sucrase activity in seed coat. Based on this, they put forward a model, in which sucrase regulates the unloading process in the early stage of seed development, and the role of cell wall sucrase is to establish the strength of the pool in the early stage.

[43]

Miller et al. (1992) came to the same conclusion when studying the genetic defect mutant of maize. In this mutant, the weight of seeds is only 1/5 of that of normal seeds, and sucrase is inactive, which interferes with the transportation of photosynthetic products to developing seeds.

In the storage organs of sugar such as fruits, the activity of high acid sucrase is closely related to the accumulation of hexose. In the wild tomato fruit mainly storing sucrose, the activity of sucrase decreased very little, but in the cultivated tomato fruit mainly storing hexose, the activity of sucrase increased at the beginning of fruit ripening [44]. Reducing the activity of sucrase by antisense or gene suppression can transform the main stored hexose in fruit into sucrose, indicating that there is a soluble sucrase in tomato, which controls the composition of sugar. Similar results were obtained in the study of potato tuber sugar stored at low temperature. Reducing the activity of sucrase by antisense means leads to the decrease of hexose/sucrose ratio, but does not change the total sugar content [45]. Therefore, soluble acid invertase is used as a regulator of sugar components. [35, 39]

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Research progress of plant sucrase

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Research progress of plant invertase

Si Lizhen, Chu Caicheng

(Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing10010/)

A summary

In higher plants, sucrose is the main source of assimilated carbon transported from the "source" tissue to the "sink" tissue, and sucrose is decomposed into glutamic acid and fructose by invertase in the "sink" tissue, which provides carbon and energy for cells and is beneficial to plant growth and development. Therefore, invertase plays an important role in source-sink interaction and plant energy supply. In this paper, the classification, main characteristics, gene regulation and function of sucrase are reviewed.

Key words: plant, invertase, regulation