ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 3, с. 426-437
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
y%K 581.1
LOW SINK-INDUCED STOMATAL CLOSURE ALTERS PHOTOSYNTHETIC RATES OF SOURCE LEAVES IN BEANS AS DEPENDENT ON H2O2 AND ABA ACCUMULATION IN GUARD CELLS1
© 2014 M. Xu***2, W. Duan*2, P. G. Fan*, B. H. Wu*, L. J. Wang*, L. Ma*,
D. D. Archbold***, S. H. Li*,****
*Institute of Botany, Chinese Academy of Sciences, Beijing, P.R. China **Graduate University, Chinese Academy of Sciences, Beijing, P.R. China ***Department of Horticulture, University of Kentucky, Agricultural Science Center North, Lexington, United Kingdom ****Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese
Academy of Sciences, Wuhan, P.R. China Received January 23, 2013
Low sink demand provided by pod removal and stem girdling of beans (Viciafaba, cv. Daqingshan) (—Sink) induced a significantly lower net photosynthetic rate (Pn), stomatal conductance (gs), internal CO2 concentration (Ci), and transpiration rate (E) compared with pod and root sink retention (CK). This depression in Pn was due to stomatal limitation. Low sink demand of—Sink plants resulted in a higher leaf sucrose content, but a lower sucrose content in guard cells. Moreover, the significant accumulation of H2O2 and ABA were observed in both leaves and guard cells of —Sink plants. The most intensive electron dense deposit of cerium perhydroxides, produced by H2O2 reaction with cerium chloride, was present in the cell walls, especially the dorsal walls of guard cells. Immunogold electron-microscopy localization of ABA showed that ABA was distributed in ventral walls of guard cells and the intercellular space of mesophyll cells of—Sink leaves in contrast to CK plants. Application of exogenous sucrose to isolated bean leaves increased H2O2 and ABA contents. H2O2 and ABA in leaves was likely generated by two independently regulated pathways, each affected by the high sucrose concentration induced by low sink demand. Increased sucrose in leaves in response to low sink demand may have caused the increase of H2O2 and ABA, and their accumulation in mesophyll cells and guard cells was likely the primary cause for stomatal closure under low sink demand.
Keywords: Vicia faba — low sink demand — photosynthesis — stomatal closure — abscisic acid — hydrogen peroxide
DOI: 10.7868/S0015330314020195
INTRODUCTION
A decline in leaf photosynthesis in response to low sink demand has been observed in many higher plants, such as peach [1], citrus [2], soybean [3], and coffee [4]. Many studies have focused primarily on carbohydrate accumulation in source leaves with evidence
This text was submitted by the authors in English. ! These authors contributed equally to this work.
Abbreviations: Ci — internal CO2 concentration; CK — control; DPI — diphenyleneiodonium chloride; E — transpiration rate; EDC — 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide; gs — stomatal conductance; PAR — photosynthetically active radiation; PBS — phosphate-buffered saline; Pn — net photosynthetic rate; PVP — polyvinylpyrrolidone; Suc — sucrose; TCA — trichloroacetic acid; Tleaf — leaf temperature; Tun — sodium tungstate; AsA — ascorbic acid.
Corresponding author. Shaohua Li. Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, 430074 Wuhan, P.R. China. Fax. +86 27 8751-0599; e-mail. shhli@wbgcas.cn
supporting the hypothesis of end-product inhibition of photosynthesis [5]. However, some studies failed to show a relationship between carbohydrate accumulation and decreased net photosynthetic rate (Pn) caused by low sink demand [6]. Moreover, studies with peach indicated that the accumulation of end products did not reduce the potential activity of related carbon-metabolic enzymes despite a considerable decrease in Pn in response to low sink demand [7]. The specific mechanism for the effect of low sink demand on photosynthesis is still unclear, although the feedback regulation hypothesis was proposed over 150 years ago.
Over the last 20 or more years, numerous studies have demonstrated a strong positive correlation between gs and Pn under a variety of different source-sink relations [4, 8]. Our previous studies have shown that both a decreased gs and increased leaf temperature (Tleaf) have a relationship to decreased Pn in response to low sink demand [8]. We suggested that decreased gs
might be considered as the trigger or promoter and increased Tleaf as the regulator of photosynthesis under low sink demand [8]. Our studies also have shown that water outflow from fruit into leaves did not influence Pn, and the lower Pn in low sink demand was not due to water status of source leaves influenced by removing fruits [9]. High Tleaf resulted in reversible and/or irreversible damage to chloroplasts caused by low leaf transpiration under low sink demand [4].
Stomatal closure takes place when ion and solute loss or release from guard cells raises the osmotic potential or reduces the water potential causing water efflux to the apoplast [10]. ABA and H2O2 are two important factors regulating stomatal closure [11]. ABA was the most effective and common factor inducing stomatal closure by regulating ion channels to affect the K+ efflux from guard cells [12]. The effects of H2O2 on guard cells were first reported by McAinsh et al. [13]. H2O2 at high concentrations resulted in the release of water, efflux and influx of ions, and the loss of turgor, inducing stomatal closure. There would be significant value in clarifying the role of ABA and H2O2 on stomatal movement with an altered source—sink relations in order to understand the regulatory mechanisms of reduced photosynthesis under low sink demand; something few studies have done.
The objective of this study was to investigate the regulatory mechanisms of photosynthesis under low sink demand in bean plants by removing pods and girdling stems to isolate roots, in particular focusing on the possible roles of ABA and H2O2 on stomatal closure.
MATERIALS AND METHODS
Materials and treatments. Bean (Vicia faba, cv. Da-qingshan) plants were grown from seeds (Xinnong-taihua Biotechnology, Beijing, China) in 25 x 25 cm plastic plots, supplied with water and fertilizer under natural sunlight in a greenhouse. Two experiments were carried out during the 2009 growing season at the Institute of Botany, Chinese Academy of Sciences, Beijing, China.
Experiment I. Uniform two-month-old plants in the reproductive stage were chosen for source—sink manipulations. The terminal shoots of plants were removed (May 23), leaving eight mature compound leaves and eight bean pods. Three days after removal of the terminal shoots (May 26), the beans of one half of plants were removed from all eight pods, and the transport of assimilates from source leaves to the roots was blocked by horizontally girdling the base of the main stem with 10% (w/v) TCA (-Sink treatment) [14]. TCA was used on dahlia plants in the study of source-sink relations [14], and our preliminary experiments on bean plants showed that TCA application on part of the stem had non-significant effect on Pn compared with control (CK) (data not shown). This result indi-
cates that the toxic effect of TCA on photosynthesis may be ignored during a short period. At the same time, the pods on the other half of plants were left intact, and the bases of the stems were treated with 10% TCA longitudinally in order to remove the possible impact of mechanical injury (CK) (fig. 1). All the manipulations were applied at about 07:00 UT and completed in 15 min.
Five plants of each treatment (i.e., five replicates) were randomly selected for the measurement of the photosynthetic gas exchange, and five mature leaves on each of five replicate plants of both treatments (25 plants per treatment) were sampled for measurements of sucrose, H2O2, and ABA contents at the same time as photosynthesis measurements. Another four fully expanded leaves per treatment (one leaf per replicate, 20 plants per treatment) were harvested for examining the subcellular localization of H2O2 and ABA.
Experiment II. Young bean plants in a vegetative stage were used to investigate the effect of exogenous sucrose on the generation of H2O2 and ABA and a possible relationship between this compounds. Whole plants were excised about 5 cm above the ground, and quickly cut again under water solutions to prevent air entry into the vascular tissue. Three sub-experiments were carried out, and all the sub-experiments had a double-distilled water only as a control and 0.05 M sucrose (Suc) as a treatment. Two treatments, 0.01 M ascorbic acid (AsA, a H2O2 scavenger) and 0.05 M sucrose plus 0.01 M ascorbic acid (Suc + AsA) were added in sub-experiment 1 to investigate the effect of H2O2 on the generation of ABA. In sub-experiment 2, 0.01 M diphenyleneiodonium chloride (DPI, an NADPH oxidase inhibitor) and 0.05 M sucrose plus 0.01 M DPI (Suc + DPI) were added to investigate the effect of ABA on the generation of H2O2 by the pathway of NADPH oxidase. In sub-experiment 3, 2 mM sodium tungstate (Tun, an ABA synthesis inhibitor) and 0.05 M sucrose plus 2 mM Tun (Suc + Tun) were added to investigate the effect of a total inhibition of ABA biosynthesis on the H2O2 generation. Five plants of each treatment (five replicates) were randomly arranged in all the three sub-experiments, and the leaves of all of the treatments were sampled after 0, 0.5, 1, 2, 4, 6, and 8 h. The leaf sucrose, H2O2, and ABA concentrations were then measured.
Measurement of photosynthesis and relative leaf physiological responses. Photosynthetic gas exchange was measured with a Li-6400 portable photosynthesis system ("Li-Cor", United States) every 2 h from 08:00 UT (1 h after initiating the treatment) to 16:00 UT on May 26 (a clear day). Measurements were made on one fixed mature compound leaf from each of five plants per treatment
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