Two-factor ANOVA of SSH and RNA-seq analysis reveal development-associated Pi-starvation genes in oilseed rape
The 5-leaf-stage rape seedlings were more insensitive to Pi starvation than that of the 3-leaf-stage plants, which may be attributed to the higher expression levels of ethylene signaling and sugar-metabolism genes in more mature seedlings.
Traditional suppression subtractive hybridization (SSH) and RNA-Seq usually screen out thousands of differentially expressed genes. However, identification of the most important regulators has not been performed to date. Here, we employed two methods, namely, a two-round SSH and two-factor transcriptome analysis derived from the two-factor ANOVA that is commonly used in the statistics, to identify development-associated inorganic phosphate (Pi) starvation-induced genes in Brassica napus. Several of these genes are related to ethylene signaling (such as EIN3, ACO3, ACS8, ERF1A, and ERF2) or sugar metabolism (such as ACC2, GH3, LHCB1.4, XTH4, and SUS2). Although sucrose and ethylene may counteract each other at the biosynthetic level, they may also work synergistically on Pi-starvation-induced gene expression (such as PT1, PT2, RNS1, ACP5, AT4, and IPS1) and root acid phosphatase activation. Furthermore, three new transcription factors that are responsive to Pi starvation were identified: the zinc-finger MYND domain-containing protein 15 (MYND), a Magonashi family protein (MAGO), and a B-box zinc-finger family salt-tolerance protein. This study indicates that the two methods are highly efficient for functional gene screening in non-model organisms.
KeywordsEthylene signaling Pi starvation RNA-Seq Sugar metabolism Suppression subtractive hybridization (SSH)
Differentially expressed gene
Suppression subtractive hybridization
This work was supported by the National Natural Science Foundation of China (31560402 and 31770322). We thank LetPub (http://www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Chiou TJ, Lin SI (2011) Signaling network in sensing phosphate availability in plants. Annu Rev Plant Biol 62:185–206. https://doi.org/10.1146/annurev-arplant-042110-103849 CrossRefGoogle Scholar
- Datta R, Kumar D, Sultana A, Hazra S, Bhattacharyya D, Chattopadhyay S (2015) Glutathione regulates 1-aminocyclopropane-1-carboxylate synthase transcription via WRKY33 and 1-aminocyclopropane-1-carboxylate oxidase by modulating messenger RNA stability to induce ethylene synthesis during stress. Plant Physiol 169:2963–2981. https://doi.org/10.1104/pp.15.01543 Google Scholar
- Jain A, Poling MD, Karthikeyan AS, Blakeslee JJ, Peer WA, Titapiwatanakun B et al (2007) Differential effects of sucrose and auxin on localized phosphate deficiency-induced modulation of different traits of root system architecture in Arabidopsis. Plant Physiol 144:232–247. https://doi.org/10.1104/pp.106.092130 CrossRefGoogle Scholar
- Lei M, Zhu C, Liu Y, Karthikeyan AS, Bressan RA, Raghothama KG et al (2011b) Ethylene signalling is involved in regulation of phosphate starvation-induced gene expression and production of acid phosphatases and anthocyanin in Arabidopsis. New Phytol 189:1084–1095. https://doi.org/10.1111/j.1469-8137.2010.03555.x CrossRefGoogle Scholar
- Liang P, Averboukh L, Keyomarsi K, Sager R, Pardee AB (1992) Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells. Cancer Res 52:6966–6968Google Scholar
- Lopez-Bucio J, Hernandez-Abreu E, Sanchez-Calderon L, Nieto-Jacobo MF, Simpson J, Herrera-Estrella L (2002) Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiol 129:244–256. https://doi.org/10.1104/pp.010934 CrossRefGoogle Scholar
- Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, Bligny R et al (2005) A genome-wide transcriptional analysis using Arabidopsis thaliana affymetrix gene chips determined plant responses to phosphate deprivation. Proc Natl Acad Sci USA 102:11934–11939. https://doi.org/10.1073/pnas.0505266102 CrossRefGoogle Scholar
- Mita S, Murano N, Akaike M, Nakamura K (1997) Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for beta-amylase and on the accumulation of anthocyanin that are inducible by sugars. Plant J 11:841–851. https://doi.org/10.1046/j.1365-313X.1997.11040841.x CrossRefGoogle Scholar
- Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394CrossRefGoogle Scholar