Effects of different light quality on growth, photosynthetic characteristic and chloroplast ultrastructure of upland cotton (Gossypium hirsutum L.) seedlings

doi: 10.9755/ejfa.2016-10-1387


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Light affected the growth and development of plants (Gong et al., 2015). Two main light qualities 33 were detected by pigment systems in plants, phytochrome and blue-absorbing pigments (BAPs). 34 Phytochrome was most sensitive to red light and far-red light, while BAPs are influenced by B and 35 ultraviolet-A (UV-A) light spectrum (Moe and Heins, 1990). Both light quantity of photon flux 36 and wavelength were very important for plant growth and development (Moshe and Dalia, 2007). 37 R LEDs light might increase starch accumulation by inhibiting the translocation of photosynthates 38 out of leaves (Saebo et al., 1995). In addition, B LEDs light were important for the formation of 39 chlorophyll, chloroplast development and stomatal opening (Senger et al., 1982). Light from B 40 plus R LEDs influenced anatomical features, photosynthesis, growth and development in pepper, 41 lily, strawberry, cherry tomato, rapeseed, non-heading Chinese cabbage and chrysanthemum 42 (Schuerger et al., 1997;Lian et al., 2002;Nhut et al., 2003;Kim et al., 2004;Kurilcik et al., 2008;43 Liu et al., 2011a43 Liu et al., , 2011bFan et al., 2013a;2013b;Li et al., 2013). 44 LEDs were solid-state, long-lasting and durable sources of narrow-band light which can be used in 45 a range of horticultural and photo-biological applications. LEDs provided an opportunity to 46 optimize the spectra for a given plant response and have been used as primary light sources for 47 4 seedlings were assigned to each light treatment according to the same light intensity; The 89 experiment was completely randomized design with three replications. Seedlings were treated by the 90 type's lights for 40 days. The spectral-energy distribution of the BR (1:8, 1:3, 1:1, 3:1, 11W) 91 LEDs, B LEDs (11W), R LEDs (11W), and FL (T5, 28W) was measured using an spectral 92 photometer (OPT-2000, Optpeco Inc., Beijing, China; Fig 1). The BR LEDs was determined 93 according to the proportion of total light intensity. 94 Assessment of morphological index 95 The plants were harvested 40 days after applying treatment. Seedlings were dried at 85°C until a 96 constant mass was reached to determine dry mass. The mass of each seedling was measured using 97 an electronic balance. Stem length was measured from the main stem base to the top of a seedling 98 using a ruler, and stem diameter was measured at the internode nearest to the root using a Venire 99 caliper. The leaf area (in cm 2 ) of each seedling was measured using aLeaf Area Meter (LI-3000, 100 LI-COR Inc., USA). 101

Assessment of chlorophyll content
102 Chlorophyll was extracted from the leaves of fifteen seedlings at a similar position within each 103 treatment to examine chlorophyll content. The fresh leaves were weighed to 0.1 g. 0.1 g leaf 104 samples were placed into a mortar with quartz sand, and 10 mL of 80% acetone was added. The 105 chlorophyll was then extracted until the leaf turned white. The optical density (OD) was measured 106 using a spectrophotometer (UV-1200, Jin Peng Inc., Shanghai, China) The photosynthetic rate was performed with a portable photosynthesis system (LI-6400, LI-COR  119 Inc., Lincoln, USA) from 9:30-10:30 am. PPF was set to measure at 100 μmol m -2 · s -1 , and the 120 experimental conditions such as leaf temperature, CO 2 concentration and relative humidity were 121 24 ± 2°C, 380 ± 5 μL/L, and 40 ± 5%, respectively (Zeng et al., 2012). 122

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A root sample was excised from the lateral roots of fifteen seedlings at a similar position within 124 each treatment. The 0.5 g fresh sample was treated in 5 mL of 0.1% 2, 3, 5-triphenyltetrazolium 125 chloride (TTC) and 5 mL of 0.067 M potassium phosphate buffer, mixed thoroughly and kept for 126 two hours at 37°C. The reaction was terminated with 2 mL of 1 M H 2 SO 4 . Then the root was 127 removed, rinsed two to three times with distilled water, placed into a mortar with quartz sand and 128 10 mL of acetone and ground until it turned white. To make a standard curve, 50, 100, 150, 200 or 129 250 μL of 0.1% TTC was added to five volumetric flasks, and Na 2 S 2 O 4 and distilled water were 130 added to reach a volume of 10 mL. The optical density was measured using a UV-1200 131 spectrophotometer (UV-1200, Jin Peng Inc., Shanghai, China) at 490 nm. Root activity was 132 determined using the following equation: 133 Root activity (mg· g -1 h -1 ) = ρ V / W T (Li et al., 2010). Where ρ is optical density, V is volume 134 (mL) of acetone extract, T is the time (h) of reactions, and W is fresh mass (g) of the sample. 135

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Total sugar content was extracted using the method of Martin (Martin et al., 2000) with slight 137 modifications. Leaves (0.5 g) were ground in a mortar with liquid nitrogen. Then 1 mL of 80% 138 ethanol was added, and the mixture was filtered through filter paper. The filtrates were recovered, 139 and the residues were washed again with 70% ethanol and filtered. Both filtrates were mixed, and 140 then 3 mL of distilled water was added. The extract was centrifuged at 12, 000r for 15 min, and 1 141 mL of supernatant was collected. Soluble sugar was determined by the sulfuric acid-anthrone 142 method and measured at 620 nm. Sucrose was determined by the phloroglucinol method and 143 measured a UV-1200 spectrophotometer (UV-1200, Jin Peng Inc., Shanghai, China) at 480 nm. 144 The method of Takahashi (Takahashi et al., 1995) was used for starch extraction. The residue 145 obtained after ethanol extraction was re-suspended with 0.1 M sodium acetate buffer (pH 4.8) and 146 boiled for 20 min. The gelatinized starch was digested with amyloglucosidase for four hours at 147 37°C and boiled again to stop the enzymatic reaction. After cooling, the mixture was centrifuged, 6 and the amount of soluble sugar in the supernatant was determined by anthrone colorimetry. The 149 starch content was estimated by converting glucose to starch equivalents using a factor of 0.9 (Li 150 et al., 2010). 151

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Leaf sections (1 cm 2 ; 1 cm × 1 cm) including small lateral veins from fully expanded leaves (from 153 the second or third nodes) of fifteen seedlings were selected and fixed for two days in 50 mL of 154 formaldehyde-based fixative solution containing 95% ethanol, glacial acetic acid and 37% 155 formaldehyde (95:5:5, v / v / v). The leaf samples were dehydrated in a graded ethanol series (75%, 156 85%, 95%, 100%, and 100%), embedded in paraffin, sectioned, mounted on glass slides, and 157 treated with safranin and fast green stain (Li et al., 2010). The stained sections of leaf tissues were 158 analyzed using a microscope (DP71, Olympus Inc., Japan). Images were viewed on a monitor and 159 analyzed using Motic Images Plus 2.0. Leaf cross-sections were measured for leaf thickness, 160 lengths of palisade tissue and spongy tissue. 161

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Samples of fifteen seedlings were collected from fully expanded leaves (from the second or third 163 nodes) of each seedling for stomatal observation. Absorbent cotton fiber was wetted with water, 164 and the abaxial and adaxial surfaces of the leaves were swabbed. When the leaf was dry, 165 transparent nail polish was brushed onto both sides. When the nail polish had air dried and formed 166 a membrane, transparent adhesive tape was pressed onto both sides of each leaf, stripped, and then 167 pressed on a slide. The slide was treated with a neutral plastic seal to make a temporary slide. 168 Epidermal fingerprints were observed by using an optical microscope (Li et al., 2010). The slides 169 were analyzed using an Olympus microscope (DP71, Olympus Inc., Japan). The area and 170 frequency of stomata were measured using Motic Images Plus 2.0. 171

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Fully expanded leaves from fifteen seedlings at a similar position within each treatment were 173 destructively sampled. Leaf sections (0.25 cm 2 ; 0.5 cm × 0.5 cm) including small lateral veins 174 were excised from the second leaf from the top and about 1 cm away from the petiole base. The 175 samples were fixed in 2.5% glutaraldehyde (pH 7.0) at 4°C for 12 hours and bled by vacuum 176 pump. The leaf sections were rinsed three times with 0.1 M phosphate buffer for 20 min, fixed in 177 1% osmic acid (pH 7.0) for two hours, rinsed three times with 0.1 M phosphate buffer for 20 min, 7 and then dehydrated in an ethanol series (30%, 50%, 70%, 80%, 90%) for 20 min each. The 179 dehydrated samples were imbued with acetone and embedded in Epon-812 epoxy resin, 180 polymerized, sectioned, and stained with 2% uranyl acetate solution (pH 4.2) for 30 min followed 181 by lead citrate solution (pH 12) for 30 min. The stained samples were then rinsed with distilled 182 water 30 times. After the sections were dry, the samples were observed using a transmission 183 electron microscope (H-7650, Hitachi Inc., Japan). 184

Statistical Analyses 185
Statistical analyses were conducted using SPSS statistical product and service solutions for 186 windows version 18.0 (SPSS, Chicago, USA). Each treatment was replicated three times. The data 187 were analyzed using an analysis of variance (ANOVA), and the differences between means were 188 tested using Duncan's multiple range test (P＜0.05). 189

Changes in growth and chlorophyll contents under different blue plus red LEDs lights 191
The measured parameters of the growth and morphogenesis of seedlings, including fresh weight, 192 dry weight, root length, stem length, stem width and leaf area, showed differences under the four 193 mixtures of blue plus red LEDs. Fresh weight, dry weight, root length, stem length, stem width 194 and leaf area were the greatest in the seedlings grown under the BR1:8 LEDs compared with the 195 other lights (Table 1). The present results demonstrate that the BR1:8 LEDs provided more 196 suitable light for the growth of upland cotton seedlings than the other ratios of blue plus red LEDs 197 (BR1:3, 1:1 or 3:1). The chlorophyll a, chlorophyll b and total chlorophyll contents were the 198 greatest in the seedlings grown under BR1:8, which showed significantly higher than the BR3:1, 199 1:1 or 1:3; However, the BR3:1, 1:1 or 1:3 lights had no significant drfferences (Table 2). 200

Changes in growth parameters under different lights 201
Different lights had variable effects on the growth of upland cotton seedlings. Compared with FL, 202 fresh mass, dry mass, root length and stem width were significantly greater in seedlings grown 203 under BR1:8 LEDs; Stem length and leaf area were significantly greater in seedlings grown under 204 R LEDs (Table 3). 205

Changes in root activity under different lights 206
Different lights varied significantly in their effects on root activity in upland cotton seedlings. 207 Root activity of seedlings grown under BR1:8 LEDs was 95.26 mg · g -1 h -1 , a value 28.21% higher 208 8 than those of the seedlings grown under B LEDs. Root activity was the lowest in seedlings grown 209 under FL (Fig. 2). 210

Changes in leaf chlorophyll contents under different lights 211
The trend of chlorophyll a, chlorophyll b and total chlorophyll contents under four treatments were 212 the same, the values were highest in seedlings under B LEDs, followed by those grown under 213 BR1:8 LEDs, and the lowest in those grown under FL (Fig. 3). However, contents of chlorophyll a, 214 chlorophyll b and total chlorophyll of seedlings under BR1:8 LEDs and R LEDs were not 215 statistically different. 216

Changes in photosynthetic rate under different lights 217
Different lights exhibited obvious differences in their effects on photosynthetic rate of upland 218 cotton seedlings. Photosynthetic rate of seedlings grown under BR1:8 LEDs was 8.19 μ mol m -2 219 s -1 , a value 44.9% higher than those of the seedlings grown under FL. Compared with FL, 220 photosynthetic rate of seedlings grown under B LEDs were the greatest. However, photosynthetic 221 rate of seedlings grown under BR1:8 LEDs and B LEDs were not statistically different (Fig. 4). 222

Changes in photosynthetic production under different lights 223
The trend of sucrose, soluble sugar and starch concentrations under four treatments were the same, 224 the values were highest in seedlings under R LEDs, followed by BR1:8 LEDs and the lowest in 225 those grown under FL (Table 4). 226

Changes in leaf anatomy under different lights 227
Leaf thickness and spongy tissue length were the greatest in seedlings grown under B LEDs, 228 followed by those grown under BR1:8 LEDs, and the smallest in seedlings grown under FL. Leaf 229 thickness of seedlings under R LEDs and FL were not statistically different. Length of palisade 230 tissue was the greatest in seedlings grown under BR1:8 LEDs. However, leaf thickness of 231 seedlings under B and R LEDs were not statistically different. (Table 5). 232

Changes in chloroplast ultrastructure under different lights 233
Seedlings grown under BR1:8 LEDs exhibited a high integrity of the chloroplast ultrastructure 234 with well-developed lamellar structure, elliptical and well-developed starch grains, and thick grana 235 and lamellae (Fig. 5 B and 6 B). Seedlings grown under B LEDs also exhibited a high integrity of 236 the chloroplast ultrastructure with a clearly visible lamellar structure and elliptical starch grains 237 ( Fig. 5 C and 6 C). When seedlings grown under R LEDs, the most chloroplasts exhibited a 238 9 disrupted ultrastructure with disjoint and ruptured grana and lamellae, but the number and volume 239 of starch grains were greater than in chloroplasts of seedlings grown under the other light sources 240 ( Fig. 5 D and 6 D). When seedlings were grown under FL, the chloroplast ultrastructure was 241 substantially modified, the chloroplast lamellae were distorted, and the lamellar structure was faint 242 (Fig. 5 A and Fig. 6 A). The present results demonstrated that chloroplast structures were well 243 developed in seedlings grown under BR1:8 LEDs and B LEDs. 244

Changes in leaf stomata features under different lights 245
Stomatal areas on the adaxial and abaxial surfaces of leaves were the greatest in seedlings grown 246 under B LEDs, followed by those grown under BR1:8 LEDs, and the smallest in seedlings grown 247 under FL (Table 6 and  and thick grana and lamellae (Fig. 5 B and 6 B), seedlings grown under B LEDs also exhibited a 285 high integrity of the chloroplast ultrastructure with a clearly visible lamellar structure (Fig. 5 C  286 and 6 C). Christopher and Mullet (1994) reported that the expression of a number of 287 chloroplast-encoded genes requires high irradiance B light. Our result was consistent with the 288 previous studies. It might because that cryptochromes (CRYs) and phototropins were specifically 289 sensitive to B light, and phytochromes were specifically sensitive to R light (Whitelam and 290 Halliday, 2007). 291 The effects of spectral quality on anatomical changes in leaf tissues of pepper plants were 292 generally correlated with the amount of B LEDs light (Schuerger et al., 1997). The largest areas of 293 palisade cells had been observed in birch leaves exposed to B LEDs light (Saebo et al., 1995). The present results are inconsistent with those of Schuerger (1997) and Saebo (1995). The 299 palisade tissues contained more chloroplasts than spongy tissues, and many photosynthetic 300 pigments and enzymes were distributed in the grana and lamella of chloroplasts (Pan et al., 2008). 301 Our results also showed that the seedlings grown under the B plus R light and B light were grown 302 well. Longer palisade tissues and thicker grana lamella of the chloroplast in leaves might be 303 beneficial for the growth of upland cotton seedlings. 304 Phytochrome affected photosynthesis by affecting chlorophyll content (Casal, 2000). Our results 305 showed that the photosynthetic rate and the pigments was the highest under B LEDs, followed by 306 B:R=1:8 LEDs (Fig. 3 and 4). Meanwhile, the net photosynthetic rate of chrysanthemum was the 307 lowest under B LEDs, but with the highest pigments (Kim et al., 2004). However, the highest 308 photosynthetic pigments were in tomato leaves with R, B plus green LEDs treatment, but net  (Table 6 and Fig. 7). A direct effect of photochrome on stomatal development and 317 higher SPAD values along with higher numbers of stomata have been recorded under B LEDs 318 (Farquhar and Sharkey, 1982). In the present study, stomatal development might be affected by 319 the chlorophyll content, which is related to the stomatal area in upland cotton seedlings grown 320 under B LEDs. 321

How LEDs on photosynthetic production metabolism? 322
Light decreased the chlorophyll content of cymbidium (Tanaka et al., 1998). The lowest pigments 323 in tomato leaves of seedlings were found in those with R LEDs treatment (Liu et al., 2011a). The 324 present results demonstrated that the chlorophyll content was lower (Fig. 3) in upland cotton 325 seedlings grown under R LEDs than those grown under B LEDs or B plus R LEDs. The results are 326 consistent with those of Tanaka and Liu. Therefore, R LEDs may decrease the chlorophyll content 327 of leaves in upland cotton. 328 12 Starch was the major storage carbohydrate in plants and has many important functions (Geiger et 329 al., 1995). R LEDs light enhanced starch accumulation in Glycine and Sorghum (Britz and Sager, 330 1990). Light quality regulated the carbohydrate metabolism of higher plants, the carbohydrate 331 content was high under R LEDs light (Kowallik, 1982). The accumulation of starch in chloroplasts, 332 which was enhanced by R LEDs light, may inhibit photosynthesis. Thus, R LEDs light appeared 333 to inhibit the translocation process (Saebo et al., 1995). Excess starch accumulation inhibited 334 photosynthesis in leaves (Bondada and Syvertsen, 2005). Chloroplast of cherry tomato leaves 335 under R LEDs was relatively rich in starch granules (Liu et al., 2011b). The present results 336 demonstrated that contents of sucrose, soluble sugar and starch were greatest (Table 4) in 337 seedlings grown under R LEDs and that the number and volume of starch grains were significantly 338 increased in chloroplasts of seedlings grown under R LEDs (Fig. 6 D), the photosynthetic rate was 339 lower in seedlings grown under R LEDs compared with those grown under blue LEDs or B plus R 340 LEDs (Fig. 4).The present results were consistent with those of the previous studies (Kowallik, 341 1982;Britz and Sager, 1990;Saebo et al., 1995;Bondada and Syvertsen, 2005;Liu et al., 2011b). 342 The photosynthetic carbon metabolic pathway was not static but was influenced by environmental 343 conditions (Zhang et al., 2015). R LEDs promoted the production of photosynthetic products but 344 might inhibit the transportation of photosynthetic products out of the leaves, starch accumulation 345 in the leaves and leaf photosynthesis was prohibited in upland cotton seedlings. 346

CONCLUSIONS 347
The present study might be the first paper of determining different light qualities on growth, 348 photosynthetic characteristic and chloroplast ultra-structure of upland cotton cultivar Sumian 22. 349 The mixture blue plus red (BR1:8) LEDs light might be propitious and necessary to upland cotton 350 seedling growth and can be used as a primary lights for cotton seedling cultivation.