Identification of phloem-associated translatome alterations during leaf development in Prunus domestica L.

Abstract

Phloem plays a fundamental role in plants by transporting hormones, nutrients, proteins, RNAs, and carbohydrates essential for plant growth and development. However, the identity of the underlying phloem genes and pathways remain enigmatic especially in agriculturally important perennial crops, in part, due to the technical difficulty of phloem sampling. Here, we used two phloem-specific promoters and a translating ribosome affinity purification (TRAP) strategy to characterize the phloem translatome during leaf development at 2, 4, and 6 weeks post vernalization in plum (Prunus domestica L.). Results provide insight into the changing phloem processes that occur during leaf development. These processes included the early activation of DNA replication genes that are likely involved in phloem cell division during leaf expansion, as well as the upregulation of phloem genes associated with sink to source conversion, induction of defense processes, and signaling for reproduction. Combined these results reveal the dynamics of phloem gene expression during leaf development and establish the TRAP system as a powerful tool for studying phloem-specific functions and responses in trees.

References

[1] Carella, P. et al. Comparative proteomics analysis of Arabidopsis phloem exudates collected during the induction of systemic acquired resistance. Plant Physiol. 171, 1495-1510 (2016).

[2] Fu, Z. Q. & Dong, X. Systemic acquired resistance: turning local infection into global defense. Annu. Rev. Plant Biol. 64, 839-863 (2013).

[3] Ham, B. K. & Lucas, W. J. The angiosperm phloem sieve tube system: a role in mediating traits important to modern agriculture. J. Exp. Bot. 65, 1799-1816 (2014).

[4] Bendix, C. & Lewis, J. D. The enemy within: phloem-limited pathogens. Mol. Plant Pathol. 19, 238-254 (2018).

[5] Folimonova, S. Y. & Tilsner, J. Hitchhikers, highway tolls and roadworks: the interactions of plant viruses with the phloem. Curr. Opin. Plant Biol. 43, 82-88 (2018).

[6] Hipper, C., Brault, V., Ziegler-Graff, V. & Revers, F. Viral and cellular factors involved in phloem transport of plant viruses. Front. Plant Sci. 4, 154 (2013).

[7] Asano, T. et al. Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem. Plant J. 32, 401-408 (2002).

[8] Omid, A., Keilin, T., Glass, A., Leshkowitz, D. & Wolf, S. Characterization of phloem-sap transcription profile in melon plants. J. Exp. Bot. 58, 3645-3656 (2007).

[9] Deeken, R. et al. Identification of Arabidopsis thaliana phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments. Plant J. 55, 746-759 (2008).

[10] Gaupels, F., Knauer, T. & van Bel, A. J. A combinatory approach for analysis of protein sets in barley sieve-tube samples using EDTA-facilitated exudation and aphid stylectomy. J. Plant Physiol. 165, 95-103 (2008).

[11] Yu, Y., Lashbrook, C. & Hannapel, D. J. Tissue integrity and RNA quality of laser microdissected phloem of potato. Planta 226, 797-803 (2007).

[12] Tetyuk, O., Benning, U. F. & Hoffmann-Benning, S. Collection and analysis of Arabidopsis phloem exudates using the EDTA-facilitated method. J. Vis. Exp. 80, 51111 (2013).

[13] Serra-Soriano, M., Navarro, J. A., Genoves, A. & Pallás, V. Comparative proteomic analysis of melon phloem exudates in response to viral infection. J. Proteom. 124, 11-24 (2015).

[14] Thieme, C. J. et al. Endogenous Arabidopsis messenger RNAs transported to distant tissues. Nat. Plants 1, 15025 (2015).

[15] Collum, T. D. & Culver, J. N. Tobacco mosaic virus infection disproportionately impacts phloem associated translatomes in Arabidopsis thaliana and Nicotiana benthamiana. Virology 510, 76-89 (2017).

[16] Riesmeier, J. W., Hirner, B. & Frommer, W. B. Potato sucrose transporter expression in minor veins indicates a role in phloem loading. Plant Cell 5, 1591-1598 (1993).

[17] Ruiz-Medrano, R., Xoconostle-Cázares, B. & Lucas, W. J. Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants. Development 126, 4405-4419 (1999).

[18] Ham, B.-K. & Lucas, W. J. Phloem-mobile RNAs as systemic signaling agents. Annu. Rev. Plant Biol. 68, 173-195 (2017).

[19] Otero, S. & Helariutta, Y. Companion cells: a diamond in the rough. J. Exp. Botany, 68, 71-78 (2017).

[20] King, R. & Zeevaart, J. Enhancement of phloem exudation from cut petioles by chelating agents. Plant Physiol. 53, 96-103 (1974).

[21] Urquhart, A. A. & Joy, K. W. Use of phloem exudate technique in the study of amino acid transport in pea plants. Plant Physiol. 68, 750-754 (1981).

[22] Atkins, C. A., Smith, P. M. & Rodriguez-Medina, C. Macromolecules in phloem exudates-a review. Protoplasma 248, 165-172 (2011).

[23] Knoblauch, M. & van Bel, A. J. Sieve tubes in action. Plant Cell 10, 35-50 (1998).

[24] Turgeon, R. & Wolf, S. Phloem transport: cellular pathways and molecular trafficking. Annu Rev. Plant Biol. 60, 207-221 (2009).

[25] Dinant, S. & Kehr, J. Sampling and analysis of phloem sap. Plant Mineral Nutrients. Methods in Molecular Biology (Methods and Protocols) pp. 185-194 (Humana Press, Totowa, NJ, 2013).

[26] Reynoso, M. A. et al. Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes. Methods Mol. Biol. 1284, 185-207 (2015).

[27] Varkonyi-Gasic, E., Gould, N., Sandanayaka, M., Sutherland, P. & MacDiarmid, R. M. Characterisation of microRNAs from apple (Malus domestica’Royal Gala’) vascular tissue and phloem sap. BMC Plant Biol. 10, 159 (2010).

[28] Oparka, K. J. & Turgeon, R. Sieve elements and companion cells-traffic control centers of the phloem. Plant Cell 11, 739-750 (1999).

[29] Esau, K. The phloem. Encyclopedia of Plant Anatomy p. 505 (Gebrüder Born-traegger, Berlin-Stuttgart, 1969).

[30] Sjolund, R. & Shih, C. Freeze-fracture analysis of phloem structure in plant tissue cultures: I. The sieve element reticulum. J. Ultrastruct. Res. 82, 111-121 (1983).

[31] Fisher, D. B., Wu, Y. & Ku, M. S. Turnover of soluble proteins in the wheat sieve tube. Plant Physiol. 100, 1433-1441 (1992).

[32] Sun, Q. et al. High-efficiency and stable genetic transformation of pear (Pyrus communis L.) leaf segments and regeneration of transgenic plants. Acta Physiol. Plant. 33, 383-390 (2011).

[33] Dutt, M., Ananthakrishnan, G., Jaromin, M., Brlansky, R. & Grosser, J. Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants. Tree Physiol. 32, 83-93 (2012).

[34] Miyata, L. Y. et al. GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters. Plant Cell Rep. 31, 2005-2013 (2012).

[35] Mustroph, A. et al. Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proc. Natl. Acad. Sci. USA 106, 18843-18848 (2009).

[36] Stadler, R. & Sauer, N. The Arabidopsis thaliana AtSUC2 gene is specifically expressed in companion cells. Plant Biol. 109, 299-306 (1996).

[37] Takahashi, H. et al. The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana. Plant J. 23, 171-182 (2000).

[38] Truernit, E. & Sauer, N. The promoter of the Arabidopsis thaliana SUC2 sucrose-H+symporter gene directs expression of β-glucuronidase to the phloem: evidence for phloem loading and unloading by SUC2. Planta 196, 564-570 (1995).

[39] Verde, I. et al. The Peach v2. 0 release: high-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity. BMC Genomics 18, 225 (2017).

[40] Rubio, M. et al. Analysis of gene expression changes in peach leaves in response to Plum pox virus infection using RNA-Seq. Mol. Plant Pathol. 16, 164-176 (2015).

[41] Wang, L. et al. Deep RNA-Seq uncovers the peach transcriptome landscape. Plant Mol. Biol. 83, 365-377 (2013).

[42] Rubio, M. et al. Gene expression analysis of Plum pox virus (Sharka) susceptibility/resistance in apricot (Prunus armeniaca L.). PLoS ONE 10, e0144670 (2015).

[43] Zhao, C., Craig, J. C., Petzold, H. E., Dickerman, A. W. & Beers, E. P. The xylem and phloem transcriptomes from secondary tissues of the Arabidopsis root-hypocotyl. Plant Physiol. 138, 803-818 (2005).

[44] Lipka, E. et al. The phragmoplast-orienting kinesin-12 class proteins translate the positional information of the preprophase band to establish the cortical division zone in Arabidopsis thaliana. Plant Cell 26, 2617-2632 (2014).

[45] Müller, R., Morant, M., Jarmer, H., Nilsson, L. & Nielsen, T. H. Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiol. 143, 156-171 (2007).

[46] Ni, D. A. et al. The Arabidopsis MCM2 gene is essential to embryo development and its over-expression alters root meristem function. New Phytol. 184, 311-322 (2009).

[47] Shultz, R. W., Lee, T.-J., Allen, G. C., Thompson, W. F. & Hanley-Bowdoin, L. Dynamic localization of the DNA replication proteins MCM5 and MCM7 in plants. Plant Physiol. 150, 658-669 (2009).

[48] Criqui, M. C. et al. Molecular characterization of plant ubiquitin-conjugating enzymes belonging to the UbcP4/E2-C/UBCx/UbcH10 gene family. Plant Physiol. 130, 1230-1240 (2002).

[49] Zhou, M. et al. WRKY70 prevents axenic activation of plant immunity by direct repression of SARD1. New Phytol. 217, 700-712 (2018).

[50] Li, H., Johnson, P., Stepanova, A., Alonso, J. M. & Ecker, J. R. Convergence of signaling pathways in the control of differential cell growth in Arabidopsis. Dev. Cell 7, 193-204 (2004).

[51] Kus, J. V., Zaton, K., Sarkar, R. & Cameron, R. K. Age-related resistance in Arabidopsis is a developmentally regulated defense response to Pseudomonas syringae. Plant Cell 14, 479-490 (2002).

[52] Carviel, J. L. et al. Investigation of intercellular salicylic acid accumulation during compatible and incompatible Arabidopsis-Pseudomonas syringae interactions using a fast neutron-generated mutant allele of EDS5 identified by genetic mapping and whole-genome sequencing. PLoS ONE 9, e88608 (2014).

[53] Carella, P., Wilson, D. C. & Cameron, R. K. Some things get better with age: differences in salicylic acid accumulation and defense signaling in young and mature Arabidopsis. Front. Plant Sci. 5, 775 (2015).

[54] Yoo, S. K., Wu, X., Lee, J. S. & Ahn, J. H. AGAMOUS-LIKE 6 is a floral promoter that negatively regulates the FLC/MAF clade genes and positively regulates FT in Arabidopsis. Plant J. 65, 62-76 (2011).

[55] Hu, J.-Y. et al. miR824-regulated AGAMOUS-LIKE16 contributes to flowering time repression in Arabidopsis. Plant Cell 26, 2024-2037 (2014).

[56] Huang, T.-K. et al. Identification of downstream components of ubiquitin-conjugating enzyme PHOSPHATE2 by quantitative membrane proteomics in Arabidopsis roots. Plant Cell 25, 4044-4060 (2013).

[57] Lin, S.-I. et al. Regulatory network of microRNA399 and PHO2 by systemic signaling. Plant Physiol. 147, 732-746 (2008).

[58] Lin, S.-Y. et al. Profiling of translatomes of in vivo-grown pollen tubes reveals genes with roles in micropylar guidance during pollination in arabidopsis. Plant Cell 26, 602-618 (2014).

[59] Branco-Price, C., Kaiser, K. A., Jang, C. J., Larive, C. K. & Bailey-Serres, J. Selective mRNA translation coordinates energetic and metabolic adjustments to cellular oxygen deprivation and reoxygenation in Arabidopsis thaliana. Plant J. 56, 743-755 (2008).

[60] Juntawong, P., Sorenson, R. & Bailey-Serres, J. Cold shock protein 1 chaperones mRNAs during translation in Arabidopsis thaliana. Plant J. 74, 1016-1028 (2013).

[61] Moeller, J. R. et al. Differential accumulation of host mRNAs on polyribosomes during obligate pathogen-plant interactions. Mol. Biosyst. 8, 2153-2165 (2012).

[62] Meteignier, L.-V. et al. Translatome analysis of an NB-LRR immune response identifies important contributors to plant immunity in Arabidopsis. J. Exp. Bot. 68, 2333-2344 (2017).

[63] Petri, C., Scorza, R. & Srinivasan, C. Highly efficient transformation protocol for plum (Prunus domestica L.). Transgenic Plants pp. 191-199 (Humana Press, Totowa, NJ, 2012).

[64] Bomblies, K. Whole mount GUS staining. Arabidopsis: A Laboratory Manual pp. 243-245 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000).

[65] Du, Z., Zhou, X., Ling, Y., Zhang, Z. & Su, Z. agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res. 38, W64-W70 (2010).