| Water deficit is one of the major limitations to apple(Malus domestica Borkh.) cultivation in arid and semi-arid region of China. Thus, improving water use efficiency(WUE) of apple tree under drought stress is of great economic importance. In present study, we performed a comparative i TRAQ-based proteomics analysis with leaves of well-watered or drought-treated cv.‘Qinguan’ and ‘Naganofuji No.2’, two varieties with different WUE response to long-term drought stress, to underline the key regulatory processes and genes contributing to higher WUE behavior under drought condition for cv. ‘Qinguan’ apple. We identified and preliminarily characterized the selected genes that potentially contributed to high WUE in apple.1. Under long-term drought stress, the WUE of cv. ‘Qinguan’ apple(M. domestica Borkh.)(tolerant to drought) was enhanced, while that of cv. ‘Naganofuji No.2’ was not enhanced. In this research, we also found that compared with well-watered plants, instantaneous water-use efficiency(WUEi) of cv. ‘Qinguan’ was enhanced by long-term drought treatment, but that of ‘Naganofuji No.2’ was not improved by drought. The WUEi of cv. ‘Qinguan’ was higher than that of cv. ‘Naganofuji No.2’ under drought, mainly because of its higher net photosynthesis rate(Pn). To dissect the underlying mechanisms of high WUE phenomenon in cv. ‘Qinguan’, we performed a comparative iTRAQ-based proteomics analysis with leaves of well-watered and drought-treated cv.‘Qinguan’ and ‘Naganofuji No.2’, combining physiological characteristics analysis, to identify the main regulatory pathways and key genes helping maintaining high WUE under drought in cv. ‘Qinguan’, providing theoretical basis for breeding new apple varieties with high WUE through molecular methods and genetic engineering.We identified 4,078 proteins(2,864 proteins were quantified), of which 594 were differentially abundant between drought-treated and well-watered leaves. The majority of increased proteins were predicted to be involved in photosynthetic pathway in drought treated cv.‘Qinguan’ leaves, indicating that regulation of photosynthesis(thus maintaining higher Pn) plays key roles to maintain higher WUE for apple tree under drought stress. The main regulatory mechanisms included: the maintaining of Calvin cycle function by increasing key enzymes, stabilization of photosynthetic electron transfer chain to ensure the supply of substrate and energy that dark reaction needed. Meanwhile, the regulation of photosynthetic electron transfer chain, combining the regulation of photorespiration and reactive oxygen species scavenging capability, enable cv. ‘Qinguan’ to keep reactive oxygen species at normal level, prevent photoinhibition, and avoid the damage to photosynthetic apparatus. Thus enhanced Pn. Except the predominant role of the regulation of photosynthesis, the number of stress-related proteins was the second most among increased proteins in drought cv. ‘Qinguan’. Among these stress-related proteins, most numbers were heat shock proteins(HSPs). In addition, the response of other pathway proteins, such as signal regulatory proteins and carbohydrate metabolism proteins, to drought also helped plants cope with such stress, improve the growth, thus enhance WUE.2. The abundance of MdAGO4.1 protein was increased in drought treated cv. ‘Qinguan’ leaves. MdAGO4.1 was one number of Argonaute(AGO) gene family. AGO is a key player in all small-RNA guided gene-silencing, which plays an important role for stress tolerance in plants. We identified and characterized 16 MdAGOs in apple genome and they were divided into three clusters. We analyzed their chromosome location, exon/intron structures of gene sequences, phylogeny, and distribution of conserved motifs. The qRT-PCR results demonstrated that these genes are induced by abiotic stresses such as drought, salt, cold and ABA treatment, indicating that they are good candidates for further analysis of their activities and functions for breeding new fruit varieties with high resistance. MdAGO4.1 was thought to be a putative gene that was possibly positive regulatory for WUE in apple, combing the result of our proteome study(MdAGO4.1 protein was increased in high WUE cv. ‘Qinguan’ apple leaves by long-term drought, whose WUE was also improved by such treatment, but this protein was not enhanced in drought treated cv. ‘Naganofuji No.2’ leaves, and the WUE of cv. ‘Naganofuji No.2’ was also not increased by drought) and MdAGO4.1 gene expression analysis(the MdAGO4.1 gene expression level was increased in cv. ‘Qinguan’ apple leaves by long-term drought stress, but was not increased in cv. ‘Naganofuji No.2’ leaves, this result was consistent with proteome study, the MdAGO4.1 gene expression level was also not increased in low WUE variety cv. ‘Honeycrisp’ apple leaves by drought) in mature leaves from three varieties of apple with different WUE. Our findings provide evidence for the participation of AGO genes in plant response to stress conditions of M. domestica. Our study also serves as a framework for future functional studies of the AGO family in apple.3. The abundance of MdPYL9 protein was increased in drought treated cv. ‘Qinguan’ leaves. MdPYL9 was one number of PYR/PYL/RCAR(PYL in short) gene family. PYR/PYL/RCAR proteins were ABA receptors, and play key roles in ABA signal transduction, which plays an important role for stress tolerance in plants. We identified and characterized 14 MdPYL genes in apple genome and they were divided into three clusters. We analyzed their chromosome location, exon/intron structures of gene sequences, phylogeny, and distribution of conserved motifs. The qRT-PCR results demonstrated that these genes are induced by abiotic stresses such as drought, salt, cold and ABA treatment, indicating that they are good candidates for further analysis of their activities and functions for breeding new fruit varieties with high resistance to drought. MdPYL9 was a putative regulator for responding to drought in apple, combing the result of our previous proteomics study(MdPYL9 protein was increased in both cv. ‘Qinguan’ apple and cv. ‘Naganofuji No.2’ by long-term drought) and MdPYL9 gene expression analysis(the MdPYL9 gene expression level was also increased both in cv. ‘Qinguan’ and cv. ‘Naganofuji No.2’ leaves by long-term drought) in mature leaves from varieties of apple with different WUE. We cloned full-length sequences of MdPYL1, MdPYL5, MdPYL7 and MdPYL9. For MdPYL9, we transformed this gene to ‘GL-3’ apple. We finally obtained MdPYL9 over-expression apple lines, and performed 50 μM ABA treatments to these transgenic seedlings. Compared with control plants, the growth of MdPYL9 over-expression apple lines under 50 μM ABA was enhanced. Considering drought can cause the increase of ABA content in plant, the over-expression of MdPYL9 can enhance the growth of apple lines under drought, thus WUE. Our findings provide evidence for the participation of PYR/PYL/RCAR genes in plant response to abiotic stress conditions and can be exploited to improve the drought resistance and WUE of M. domestica. Our study also serves as a framework for future functional studies of the PYR/PYL/RCAR family in apple. |
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