Working smarter, not harder: silencing LAZY1 in Prunus domestica causes outward, wandering branch orientations with commercial and ornamental applications

Abstract

Controlling branch orientation is a central challenge in tree fruit production, as it impacts light interception, pesticide use, fruit quality, yield, and labor costs. To modify branch orientation, growers use many different management practices, including tying branches to wires or applying growth regulator sprays. However, these practices are often costly and ineffective. In contrast, altering the expression of genes that control branch angles and orientations would permanently optimize tree architecture and reduce management requirements. One gene implicated in branch angle control, LAZY1, has potential for such applications as it is a key modulator of upward branch orientations in response to gravity. Here, we describe the phenotypes of transgenic plum (Prunus domestica) trees containing an antisense vector to silence LAZY1. We found that LAZY1 silencing significantly increased branch and petiole angles. LAZY1-antisense lines also displayed ‘wandering’ or weeping branch trajectories. These phenotypes were not associated with decreases in branch strength or stiffness. We evaluated the utility of LAZY1-antisense trees for use in two planar orchard systems by training them according to super slender axe and espalier methods. We found that the LAZY1-antisense trees had more open canopies and were easier to constrain to the trellis height. This work illustrates the power of manipulating gene expression to optimize plant architecture for specific horticultural applications.

Acknowledgements

We would like to thank Srinivasan Chinnithambi and Ralph Scorza for performing plant transformations, Elizabeth Lutton for cloning the constructs, Frank Telewski for access to and assistance with his Instron, Greg Lang for help implementing planar training systems, Peter Kohler for helping with data formatting and statistical analyses, Joy Johnson and Andrew Scheil for their assistance with photosynthesis measurements and imaging, and the MSU Clarksville Research Center Farm Manager Dan Platte and his team for helping with tree planting and maintenance. Funding for this work was provided by Michigan State University, The Michigan State Horticultural Society, The United States Department of Agriculture National Institute of Food and Agriculture HATCH project 1013242 (C.A.H.), MSU AgBioResearch Project GREEEN grant GR18-03, and the Agriculture and Food Research Initiative Competitive Grants Program, grant no. WVAW-2011-04220, from the USDA National Institute of Food and Agriculture (C.D.).

Author contributions

C.A.H. and C.D. conceived the project idea. C.A.H., A.R.K., and C.D. planned the experiments. C.A.H., D.R., M.R., and A.R.K. performed experiments and analyzed data. A.R.K. and C.A.H. wrote the manuscript. C.A.H., A.R.K., and C.D. edited the manuscript.

Data availability statement

The data underlying this article are available in the article and in its online supplementary material.

Conflict of interests

Authors declare no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Warner J. Rootstock affects primary scaffold branch crotch angle of apple trees. HortScience. 1991; 26:1266-7

[2] Quinlan JD, Tobutt KR. Manipulating fruit tree structure chem-ically and genetically for improved performance. HortScience. 1990; 25:60-4

[3] Cvetkovi ćM, Ðuri ć G, Micic N. Canopy management practices in modern plum (Prunus domestica, L.) production on vigorous rootstocks. Scientific Papers Series B Horticulture. 2017; 61:117-122

[4] Dardick C, Callahan A, Horn R. et al. PpeTAC1 promotes the horizontal growth of branches in peach trees and is a member of a functionally conserved gene family found in diverse plants species. Plant J. 2013; 75:618-30

[5] Waite JM, Dardick C. The roles of the IGT gene family in plant architecture: past, present, and future. Curr Opin Plant Biol. 2021; 59:101983

[6] Hollender CA, Waite JM, Tabb A. et al. Alteration of TAC1 expres-sion in Prunus species leads to pleiotropic shoot phenotypes. Horticulture Research. 2018; 5:26

[7] Nakamura M, Nishimura T, Morita MT. Bridging the gap between amyloplasts and directional auxin transport in plant gravit-ropism. Curr Opin Plant Biol. 2019; 52:54-60

[8] Li P, Wang Y, Qian Q. et al. LAZY1 controls rice shoot grav-itropism through regulating polar auxin transport. Cell Res. 2007; 17:402-10

[9] Arai-Sanoh Y, Takai T, Yoshinaga S. et al. Deep rooting conferred by DEEPER ROOTING 1 enhances rice yield in paddy fields. Sci Rep. 2014; 4:5563

[10] Howard TP III, Hayward AP, Tordillos A. et al. Identification of the maize gravitropism gene lazy plant1 by a transposon-tagging genome resequencing strategy. PLoS One. 2014; 9:e87053

[11] Ge L, Chen R. Negative gravitropism in plant roots. Nature Plants. 2016; 2:16155

[12] Taniguchi M, Furutani M, Nishimura T. et al. The Arabidopsis LAZY1 family plays a key role in gravity signaling within sta-tocytes and in branch angle control of roots and shoots. Plant Cell. 2017; 29:1984-99

[13] Chen Y, Xu S, Tian L. et al. LAZY3 plays a pivotal role in positive root gravitropism in Lotus japonicus. JExp Bot. 2020; 71:168-77

[14] Dougherty L. et al. A single amino acid substitution in MdLAZY1A dominantly impairs shoot gravitropism in Malus. Plant Physiology. 2023; 193:1142-1160. https://doi.org/2023.04.05.535771

[15] Godbolé R, Takahashi H, Hertel R. The lazy mutation in rice affects a step between statoliths and gravity-induced lateral auxin transport. Plant Biol. 1999; 1:379-81

[16] Li D, Zhao M, Yu X. et al. Over-expression of rose RrLAZY1 negatively regulates the branch angle of transgenic Arabidopsis inflorescence. Int J Mol Sci. 2021; 22:13664

[17] Xia X. et al. The CsLAZY1  mediates shoot gravitropism and branch angle in tea plant (Camellia Sinensis) Research Square. 2021. PREPRINT ( Version 1) 1-19

[18] Loarce Y, Cabeza A, Cañas R. et al. Isolation and molecular characterisation of TtDro1A and TtDro1B genes from Triticum turgidum subspecies durum and turgidum, study of their influ-ences on seedling root angles. Plan Theory. 2022; 11:821

[19] Waite JM, Dardick C. IGT/LAZY genes are differentially influ-enced by light and required for light-induced change to organ angle. BMC Biol. 2024; 22:8

[20] Yang P, Wen Q, Yu R. et al. Light modulates the gravitropic responses through organ-specific PIFs and HY5 regulation of LAZY4 expression in Arabidopsis. PNAS. 2020; 117:18840-8

[21] Chen J, Yu R, Li N. et al. Amyloplast sedimentation repolar-izes LAZYs to achieve gravity sensing in plants. Cell. 2023; 186: 4788-4802.e15

[22] Nishimura T, Mori S, Shikata H. et al. Cell polarity linked to gravity sensing is generated by LZY translocation from statoliths to the plasma membrane. Science. 2023; 381:1006-10

[23] Furutani M, Hirano Y, Nishimura T. et al. Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control. Nat Commun. 2020; 11:76

[24] Yoshihara T, Spalding EP.LAZY genes mediate the effects of gravity on auxin gradients and plant architecture. Plant Physiol. 2017; 175:959-69

[25] Zhebentyayeva T, Shankar V, Scorza R. et al. Genetic characteri-zation of worldwide Prunus domestica (plum) germplasm using sequence-based genotyping. Hortic Res. 2019; 6:1-13

[26] Callahan AM, Zhebentyayeva TN, Humann JL. et al. Defining the ‘HoneySweet’ insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica). Horticulture Research. 2021; 8:8

[27] Reich L. Fruiting espaliers: a fusion of art and science. Arnoldia. 1999; 59:17-24

[28] Long LE, Lang GA, Whiting MD. et al. Cherry Training Systems. Corvallis, Oregon: Oregon State University; 2015:

[29] Johnson D, Eckart P, Alsamadisi N. et al. Polar auxin transport is implicated in vessel differentiation and spatial patterning during secondary growth in Populus. Am J Bot. 2018; 105:186-96

[30] Callahan AM, Dardick CD, Scorza R. Multilocation comparison of fruit composition for ‘HoneySweet’, an RNAi based plum pox virus resistant plum. PLoS One. 2019; 14:e0213993

[31] Tustin DS, Hirst PM, Warrington IJ. Influence of orientation and position of fruiting laterals on canopy light penetration, yield, and fruit quality of ‘granny smith’ apple. J Am Soc Hortic Sci. 1988; 113:693-9

[32] Salojärvi J, Smolander OP, Nieminen K. et al. Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch. Nat Genet. 2017; 49: 904-12

[33] Yoshihara T, Iino M. Circumnutation of rice coleoptiles: its rela-tionships with gravitropism and absence in lazy mutants. Plant Cell Environ. 2006; 29:778-92

[34] Zhang H, Wafula EK, Eilers J. et al. Building a foundation for gene family analysis in Rosaceae genomes with a novel workflow: a case study in Pyrus architecture genes. Front Plant Sci. 2022; 13. https://www.frontiersin.org/articles/10.3389/fpls.2022.975942

[35] Helliwell CA, Wesley SV, Wielopolska AJ. et al. High-throughput vectors for efficient gene silencing in plants. Functional Plant Biol. 2002; 29:1217-25

[36] Petri C, Scorza R, Srinivasan C. Highly efficient transformation protocol for plum (Prunus domestica L.). In: Dunwell JM, Wetten AC,eds. Transgenic Plants:Methods and Protocols. Humana Press: Totowa, NJ, 2012,191-9

[37] Collum TD, Stone AL, Sherman DJ. et al. Translatome profiling of plum pox virus-infected leaves in European plum reveals tem-poral and spatial coordination of defense responses in phloem tissues. MPMI. 2020; 33:66-77