Integrated transcriptome and proteome analysis provides insights into anthocyanin accumulation in the 1 leaves of red-leaved poplars 2 3


 The red-leaved poplar cultivars ‘Quanhong’ and ‘Xuanhong’ are bud mutations of Populus deltoides cv. ‘Zhonglin 2025’. These cultivars are valued for their beautiful shape, lack of flying catkins, and ornamental leaf colors. However, the understanding of the molecular mechanism of anthocyanin accumulation in the leaves of red-leaved poplars is still unclear. Here, we profiled the changes of pigment content, transcriptome and proteome expression in the leaves of three poplar cultivars and the results showed that the ratios of anthocyanin to total chlorophyll in both red-leaved poplars were higher than that in ‘Zhonglin 2025’, indicating that the anthocyanin was highly accumulated in the leaves of red-leaved poplars. Based on the results of integrated transcriptome and proteome analysis, 15 and 11 differentially expressed genes/proteins involved in anthocyanin synthesis were screened in ‘Quanhong’ and ‘Xuanhong’, respectively, including the CHS, F3H, and DFR genes. Among the 120 transcription factors, 3 (HY5, HYH, and TTG2), may be directly involved in the regulation of anthocyanin synthesis in both red-leaved poplars. This study screens the candidate genes involved in anthocyanin accumulation in the leaves of red-leaved poplars and lays a foundation for further exploring the molecular mechanism of leaf red coloration in red-leaved poplars.

. Among these, anthocyanins, which belong to the flavonoid family, determine the 76 color of fruits, leaves, and flowers in most plants (Miller et al., 2011); the redness of the red-leaved 77 poplar leaves is also caused by anthocyanin accumulation (Guo, 2013). Anthocyanins  analysis between transcriptome and proteomics is beneficial to the study of multi-level regulation of 117 gene expression process, which is an effective way to study the molecular mechanisms underlying 118 many biological traits (Vogel and Marcotte, 2012). In this study, we analyzed differences in mRNA and 119 protein expression using transcriptome and proteome sequencing between red-leaved poplar cultivars

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To ensure the quality and reliability of the data analyses, the raw data were pre-processed.

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Analysis of pigment content in the leaves of red-leaved poplars

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As we all know that the immediate reason for the color change of plant leaves is due to the change of 238 the content and proportion of various pigments in the leaves. In order to understand the changes of 239 pigment content in the leaves of red-leaved poplars, we first measured the contents of chlorophyll, 7 carotenoid and anthocyanin in the leaves of the three poplar species. The results showed that the 241 chlorophyll a content in 'Quanhong' was slightly higher than that in 'Zhonglin 2025', while the 242 chlorophyll a content in 'Xuanhong' was significantly lower than that in 'Zhonglin 2025' (Fig. 1B).

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The change tendency of chlorophyll b content in both red-leaved poplars was the same as that of 244 chlorophyll a, and the difference was significant (Fig. 1C). The carotenoid content of both red-leaved 245 poplars was lower than that of 'Zhonglin 2025', and the carotenoid content of 'Xuanhong' was 246 significantly lower than that of 'Zhonglin 2025' (Fig. 1D). The anthocyanin content in both red-leaved 247 poplars was significantly higher than that in 'Zhonglin 2025' (Fig. 1E). It is important that the ratios of  Table S2).

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In this study, we identified a total of 37,846 genes, including 37,271 annotated genes and 575 263 predicted new genes. We screened a total of 6,792 DEGs in 'Quanhong', of which 3,084 were 264 significantly upregulated and 3,708 were significantly downregulated; a total of 4,398 DEGs were 265 screened in 'Xuanhong', including 2,103 significantly upregulated genes and 2,295 significantly 266 downregulated genes ( Fig. 2A). Venn diagram analysis showed a total of 2,779 genes that were 267 differentially expressed in both red-leaved poplars (Fig. 2B). Altogether, many DEGs were detected in 268 the red-leaved poplars, with slightly more genes downregulated than upregulated.    (Fig. 1E), resulting in the difference between the leaf colors of the both red-leaved poplars (Fig. 1A).  Table S7).

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Differentially expressed structural genes involved in anthocyanin synthesis, and the differentially 336 expressed transcription factors identified above, were used to form a protein-protein interaction 337 network (Fig. 7). In both 'Quanhong' and 'Xuanhong', there were close interactions among the 338 structural genes that control anthocyanin synthesis. The key genes controlling anthocyanin synthesis