Cytologic, Genetic, and Proteomic Analysis of a Yellow Leaf Mutant of Sesame (Sesamum indicum L.), Siyl-1

Leaf color mutation in sesame always affects the growth and development of plantlets, and their yield. To clarify the mechanisms underlying leaf color regulation in sesame, we analyzed a yellow-green leaf mutant. Genetic analysis of the mutant selfing revealed 3 phenotypes—YY, light-yellow (lethal); Yy, yellow-green; and yy, normal green—controlled by an incompletely dominant nuclear gene, Siyl-1. In YY and Yy, the number and morphological structure of the chloroplast changed evidently, with disordered inner matter, and significantly decreased chlorophyll content. To explore the regulation mechanism of leaf color mutation, the proteins expressed among YY, Yy, and yy were analyzed. All 98 differentially expressed proteins (DEPs) were classified into 5 functional groups, in which photosynthesis and energy metabolism (82.7%) occupied a dominant position. Our findings provide the basis for further molecular mechanism and biochemical effect analysis of yellow leaf mutants in plants.


Introduction 23
Sesame (Sesamum indicum L.) is a valuable crop with 45-63% oil in its decorticated seeds, 24 which also contain numerous beneficial minerals, antioxidants, and multi-vitamins [1]. 25 Compared with other oilseed crops, sesame is still a low yield crop with low harvest index. 26 The key research objectives in sesame are to increase the photosynthesis efficiency and the 27 yield per square area. 28 Leaf color is an important trait related with the chlorophyll content, and always affects the 29 photosynthesis efficiency and the final productivity [2]. In plants, the chloroplast structure There is a rich diversity of leaf color mutation types in plants [11][12][13][14]. 37 Leaf color mutants were divided into five types according to the color classification albino, 38 yellowing, light-green, stripe, spot, etc [15,16]. Falbel [12] divided the chlorophyll mutant 39 into two categories: (1) mutants that lack chlorophyll b, such as arabidopsis mutants [17], and 40 (2) mutants with reduced synthesis of total chlorophyll and chlorophyll b; currently most 41 mutants belong to the latter category. Leaf color characteristics controlled by both nuclear accounts for a small proportion of the leaf color mutants [20]. There are very few leaf color 48 mutations that are of the nucleo-cytoplasmic interaction type [17]. 49 At present, the research on leaf colors is focused on genetic analysis [11,[18][19][20]. This 50 method has been currently used in the study of environment stress on plants, regulation 51 mechanism of leaf color, etc. [21][22][23]. Till date, there is no study reporting the use of 52 proteomics in understanding sesame leaves color, but this method is a powerful approach to 53 identify and isolate different proteins. 54 In this study, we generated a yellow color sesame mutant, Siyl-1, using EMS mutagenesis. 55 The mutant exhibited a stably inherited yellow leaf trait in different years and growing 56 environment. Furthermore, the mutant selfing will separate out three different color types: 57 light-yellow (lethal), yellow-green and normal green.

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In order to systematically analyze and parse the mutant molecular regulation mechanism, 59 we proposed to investigate the cytology, genetics, and proteomics of the sesame mutant using The yellow leaf mutant Siyl-1 was used for leaf color trait analysis (Fig. 1 To explore the genetic characteristics of the leaf color trait in the mutant Siyl-1, the 80 self-pollution and the cross hybridization of 6 groups were performed using the above 81 materials during 2014-2016 (Table 1). The leaf color trait in each sample was observed three 82 times after germination. Chi-square tests (P = 0.05) were used to determine the segregation 83 significance for the leaf color characteristic.

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To perform proteomics analysis, the healthy seeds of the three genotypes of the mutant where V is the extracted volume (mL), N is the dilution factor, and W is the fresh sample 101 weight.

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Duncan's multiple-range test was applied to analyze the chlorophyll content.

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Samples were rinsed with 0.1 mol· L -1 phosphate buffer (pH 7.2), fixed in 1% osmium acid 108 solution, and then rinsed using the same buffer.   interaction of DEPs, a protein-protein interaction network (PPI) was predicted using the online 146 analysis tool STRING 9.0 (http://string-db.org).
147  sample were performed and three technical replicates were analyzed for each RNA sample.

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SiTUB was used as an internal reference gene to normalize the relative gene expression [29].

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The relative gene expression was calculated using Pfaffl method [30]. The primer sequences 170 of the genes of DEPs for qRT-PCR are shown in Table 3.
171 Table 3 Primer sequences used for qRT-PCR of genes involved in photosynthesis and energy metabolism in To clarify the genetic background of yellow leaf color trait in mutant Siyl-1, we investigated 177 the phenotypes of the self-pollinated progeny and 6 test F 1 progeny from reciprocal cross 178 between the mutant type (Yy) and 3 wild type (yy) of Siyl-1 ( Fig. 1 and Table 1). There were 179 three phenotypes in self-pollinated progeny, i.e., light-yellow (YY), yellow-green (Yy), and 180 normal green (yy) with the expected separation ratio of 1 (YY): 2 (Yy): 1 (yy) using χ 2 tests.

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The Yy with yellow-green leaf could complete the whole life cycle. However, the YY always 196 Table 4 Chlorophyll content of Siyl-1 progeny at cotyledon stage Subsequently, we compared the ultra-structure of leaf cells in the three genotypes (Fig. 2).

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In YY, there were no obvious difference in the location of chloroplast, but their shape changed 200 ( Fig. 2A and 2B). The shape of chloroplast changed from a convex lens like shape to a 201 circular type. The lamellar structure was unclear and disordered as a loose strip. The thylakoid 202 volume dropped and the osmiophilic granules increased. In contrast, chloroplasts in Yy type 203 was larger and had a thicker spindle shape with a cavity. Meanwhile, gaps were observed in 204 the stacking and folding lamellar structure. The chloroplast structure was abnormal and the 205 osmiophilic granules appeared ( Fig. 2C and 2D). As for yy progeny (used as control), 206 chloroplasts exhibited a normal spindle shape without cavities ( Fig. 2E and 2F). The lamellar 207 structure was clear and folded tightly. Starch grains were absent; while a small amount of 208 osmiophilic granules were present in cells ( Fig. 2E and 2F). identified by comparing the two mutants with the wild type (Fig. 4). In these proteins, the 219 expression abundance of 57 proteins changed among YY/yy, and 17 among Yy/yy. The 98 220 protein spots were digested with trypsin and analyzed by MALDI-TOF-TOF analysis. Then, 221 these proteins were ultimately screened using the NCBI nr database (Fig. 3) Table 1).
233 map provides an overview of these DEPs (Fig. 6) possessed four different molecular mass or isoelectric point (Supplemental Table 1).

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Interaction of DEPs 245 STRING 9.0 was used to analyze the protein-protein interaction and molecular function of 246 DEPs (Supplemental Fig. 1, 2). The results revealed that these proteins formed a complicated 247 interaction network, and most of the core interacting proteins were related with 248 photosynthesis and energy metabolism (Supplemental Table 2). In addition, there are 21 identified involved in photosynthesis and energy metabolism (Fig. 7). The qRT-PCR was 259 used to analyze the expression levels of these genes (Fig. 7). There were 2 different gene 260 expression patterns compared with the protein expression (Supplemental Table 1   LHCs (20-24) were significantly decreased in YY when compared to the wild type plants. 331 Therefore, we speculate that those different proteins are related to the ability of capturing light 332 in Siyl-1.

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The    Table 3. Primer sequences used for qRT-PCR of genes involved in photosynthesis and energy metabolism in 588 Siyl-1 589 Table 4. Chlorophyll content of Siyl-1 progeny at cotyledon stage 590 Table 5. Proteomics comparison of the mutant types (YY and Yy) and the wild type (yy) of Siyl-1 using 2D gels

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Standard deviation was expressed by error bars among three independent replicates. A, oxygen-evolving enhancer protein