ABSTRACT
Phytoplasmas parasitize plant phloem tissue and cause many economically important plant diseases. Jujube witches'-broom disease is a destructive phytoplasma disease of Chinese jujube (Ziziphus jujuba). To elucidate the influence of phytoplasma on host photosynthetic, carbohydrate and energy metabolisms, four types of jujube tissues showing disease symptoms with different severity were investigated at the structural, physiological, and molecular levels. Quantitative real-time PCR and high-performance liquid chromatography results showed that the down-regulation of genes related to photosynthesis and the lower contents of chlorophyll in diseased leaves. This clearly inhibited the light-harvesting and photosystem II activity of photosynthesis; however, overexpression of genes related to starch, sucrose and glucose synthesis led to higher contents of these carbohydrates. Meanwhile, transmission electron microscopy images revealed that dense amounts of phytoplasmas accumulated in the sieve elements of diseased petiole phloem, and the structure of the grana and stroma lamellae of chloroplasts in the diseased leaves was destroyed. Phytoplasma infection inhibited photosynthesis and led to abnormal carbohydrate accumulation in the diseased leaves. Furthermore, comparative metabolite analysis indicated that phytoplasma infection also stimulated amino acids and energy metabolisms of the diseased leaves. Continually inhibiting the photosynthetic process and stimulating carbohydrate and energy metabolisms of diseased trees may exhaust their nutrients. Our results highlight the importance of changing host metabolisms during the pathogenic process.
Subject(s)
Carbohydrate Metabolism , Energy Metabolism , Photosynthesis , Phytoplasma/pathogenicity , Plant Diseases/immunology , Ziziphus/immunology , Chlorophyll/metabolism , Chloroplasts/ultrastructure , Models, Biological , Plant Diseases/microbiology , Plant Leaves/immunology , Plant Leaves/microbiology , Plant Leaves/physiology , Plant Leaves/ultrastructure , Plant Stems/immunology , Plant Stems/microbiology , Plant Stems/physiology , Plant Stems/ultrastructure , Ziziphus/microbiology , Ziziphus/physiology , Ziziphus/ultrastructureABSTRACT
Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1γ) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions.
