ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 2, с. 197-206
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
LOW NIGHT TEMPERATURES INHIBIT GALACTINOL SYNTHASE GENE EXPRESSION AND PHLOEM LOADING IN MELON LEAVES DURING
FRUIT DEVELOPMENT1 © 2014 J. H. Hao*, R. Yang*, J. L. Wang*, Q. Zhang*, T. L. Li**
* Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology,
Beijing University of Agriculture, Beijing, P. R. China ** Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture,
Shenyang Agricultural University, Shenyang, P. R. China Received January 11, 2013
Low night temperatures seriously affect plant growth and fruit quality. To investigate the effect of low night temperatures on the expression of galactinol synthase genes (GOLS) and phloem loading of raffinose family oligosaccharides, particular stachyose and raffinose (RFO represents stachyose and raffinose in this paper) and to gain a better understanding of the relationship between the phloem loading of RFO and fruit development, melon (Cucumis melo L.) plants at the fruit development stage were treated with temperatures of 28/12°C or 28/9°C (day/night) with 28/15°C as the control. Both the CmGOLS1 and CmGOLS2 gene expression and the activity of galactinol synthase were clearly repressed after treatments with 9 and 12 °C at night, and the effect of 9°C was more obvious. Furthermore, low night temperatures inhibited photosynthesis and caused the lower amounts of sucrose to supply the RFO synthesis. However, the total soluble sugar, RFO, and sucrose contents were increased in leaves subjected to low night temperatures. It is supposed that low night temperature blocked symplastic phloem loading, which led to the accumulation of RFO in the leaf cells. With increasing content of RFO in the leaves, the expression of GOLS genes was inhibited according to the principle of feedback, and therefore the decreased expression of GOLS limited RFO synthesis and was indirectly harmful to phloem loading, thereby affecting fruit development.
Keywords: Cucumis melo — phloem loading — CmGOLS1 and CmGOLS2 — transcription level — RFO accumulation —fruit development
DOI: 10.7868/S0015330314020055
INTRODUCTION
Low temperature is one of the most common environmental stresses for plants and potentially causes severe losses to agriculture. It induces a sequence of morphological, biochemical, and molecular alterations that negatively affect growth and productivity of plants [1], fruit vegetables in particular. During the growth of vegetable fruits, a low temperature usually not only limits plant and fruit growth but also affects fruit quality [2].
Cucumis melo L. (Cucurbitaceae) is an important vegetable prized in the world for its fruits. It is adapted
1 This text was submitted by the authors in English.
Abbreviations: DAP - days after pollination; GOLS - galactinol synthase; Pn - net photosynthetic rate; RFO - raffinose family oligosaccharides; CC - companion cells; SE - sieve elements. Corresponding author: T.L. Li. Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture, Shenyang Agricultural University, No. 120 Dong Lin Road, Shenhe District, Shenyang, P. R. China. Fax: +86-108848-7166; e-mail: haojinghongz'@aliyun.com
to warm environment and is hence sensitive to chilling temperatures. When cultivated in the winter or early spring, it is usually subjected to low night temperatures in greenhouses; the temperature is usually above 25°C during the daytime due to sunlight but is at 12° C or lower at night. Hence, a low night temperature is one of the key limitations for melon production in greenhouses during their cultivation in winter. Under these stress conditions, fruit development is impaired to some extent, including slow fruit expansion and low carbohydrate accumulation.
It is well known that fruit development is largely dependent upon the supply of photoassimilates that are imported from the leaves into the fruit via the phloem. Up to 80% of the carbon that is fixed in mature leaves during photosynthesis is exported to the heterotrophic sinks to enable their growth and development. The first step in the transport pathway is phloem loading, which plays an important role in transporting sugars to the fruit. The capability of phloem loading can represent the amount of transported
sugars to enter the phloem system, and it provides for the driving force for nutrient transport by generating turgor pressure in the long-distance conducting cells of the source organs [3].
Phloem loading is the transfer of photoassimilates from the mesophyll cells to the sieve elements (SE) and companion cells (CC) in minor veins [4]. Two ways of loading, e.g., apoplastic and symplastic, are known. At apoplastic phloem loading, sucrose enters the cell wall space and is driven across the plasma membranes of CC and/or SE by specific transport proteins [5]. At symplastic phloem loading, sucrose or other sugars are loaded into the phloem via plas-modesmata by a passive or active process. In many plant species, such as arabidopsis, tomato, soybean, maize, sugar beet, or tobacco, assimilated CO2 is exported exclusively in the form of sucrose. Sucrose can be loaded into phloem via the apoplast or symplast by a passive process, in which the concentration gradient between the leaf mesophyll and the phloem tissue drives the thermodynamically favorable movement of carbohydrates from cell to cell via the abundant plas-modesmatal connections [6]. However, in addition to sucrose, raffinose and stachyose (primary carbohydrate belonging to raffinose family oligosaccharides (RFO)) are used for long-distance transport in Cucur-bitaceae [7]. The phloem loading of RFO is completed in a symplastic manner by an active process, and the most feasible mechanism is the polymer trapping [8]. When sucrose reaches specialized CC that are sym-plastically connected to photosynthetic cells, which are known as intermediary cells and are only present in the minor veins, sucrose is converted to either raffi-nose or stachyose (oligosaccharide polymers of sucrose), which are assumed to be too large to diffuse back through the plasmodesmata.
Raffinose and stachyose are synthesized from sucrose by the sequential addition of galactose moieties that are donated by galactinol. Galactinol synthase (GOLS; EC 2.4.1.123) catalyzes the synthesis of galactinol from UDP-galactose and myo-inositol; GOLS thus plays an important regulatory role in carbon partitioning between sucrose and RFO. Galactinol synthase is immunolocalized to the intermediary cells of minor veins in cucurbits [9]. Several previous studies have shown that the C. melo CmGOLSl gene is activated in the smallest veins of the mature leaves of tobacco and arabidopsis, suggesting that the expression pattern of GOLS may be consistent with a role in RFO synthesis and phloem loading in cucurbit species as well.
Previous studies have determined the ways of phloem loading and their possible mechanisms, including RFO synthesis and phloem loading in RFO-transport-ed plants; the relationship between the expression of GOLS and RFO synthesis and phloem loading is also understood. However, little is known about the effects that low temperatures exert on GOLS expression and
the phloem loading of RFO in maturing leaves during the fruit development of Cucurbitaceae plants, and the carbohydrate source-sink connection in stachyose-transporting species, such as melon, is not well characterized, especially at low night temperatures.
Therefore, we have aimed to determine the effect of low night temperatures on GOLS transcription level, the phloem loading of RFO, and the function of GOLS expression in this process. Furthermore, we have analyzed the relationship between phloem loading and melon fruit development. This knowledge is of primary importance for understanding how symplastic phloem loading can operate under a low night temperature, thereby regulating the phloem loading of RFO to promote fruit development.
MATERIALS AND METHODS
Plant material and culture conditions. Seeds of Cu-cumis melo L., cv. Yumeiren, a popular melon variety from Northeast China, were sown in a sand/soil/peat (1 : 1 : 1, v/v) mixture and cultivated under glass greenhouse conditions (12 h light, 300-1300 ^mol/(m2 s), 12 h dark; 26 ± 2°C during the day and 15 ± 2°C at night, 50-70% relative humidity) for 30 days. The seedlings were transferred into 25 x 25 cm plastic buckets containing a mixture of organic matter of soil with NPK fertilizer (2 : 1, v/v) and were maintained in the controlled environment described above. The plants were watered and fertilized by normal cultivation management. The female flowers were hand-pollinated and tagged at anthesis, and the fruit load was limited to one per plant at the 12th to 14th node.
Low night temperature stress treatments. Following pollination for 7 days and after the young fruit had started to expand, the plants were environmentally hardened in a controlled environmental chamber to receive different night temperature treatments for 18 days (12 h light; 06:00-10:00 (475 ± 50) ^mol/(m2 s), 10:00-16:00 (1045 ± 50) ^mol/(m2 s), 16:00-18:00 (570 ± 50) ^mol/(m2 s); 26°C; 12 h dark at 15, 12, or 9°C; 15°C was used as the control, with 60% relative humidity). The low night temperature treatments were considered to initiate at day 0 (7 days after pollination (DAP)).
The leaves were collected at 0, 3, 6, 9, 12, 15, and 18 days after treatments, between 08:00 UT and 09:00 UT, and frozen in liquid nitrogen for analyzing the total soluble sugar contents and for the northern blot analysis. The fruits were harvested at 0, 3, 6, 9, 12, 15, and 18 days after treatments, between 08:00 UT and 09:00 UT Five fruits were selected at random from plants in each treatment and weighed using an analytical balance, and three fruits were frozen in liq
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