Opposing influences of TAC1 and LAZY1 on Lateral Shoot Orientation in Arabidopsis

Abstract

TAC1 and LAZY1 are members of a gene family that regulates lateral shoot orientation in plants. TAC1 promotes outward orientations in response to light, while LAZY1 promotes upward shoot orientations in response to gravity via altered auxin transport. We performed genetic, molecular, and biochemical assays to investigate possible interactions between these genes. In Arabidopsis they were expressed in similar tissues and double mutants revealed the wide-angled lazy1 branch phenotype, indicating it is epistatic to the tac1 shoot phenotype. Surprisingly, the lack of TAC1 did not influence gravitropic shoot curvature responses. Combined, these results suggest TAC1 might negatively regulate LAZY1 to promote outward shoot orientations. However, additional results revealed that TAC1-and LAZY1 influence on shoot orientation is more complex than a simple direct negative regulatory pathway. Transcriptomes of Arabidopsis tac1 and lazy1 mutants compared to wild type under normal and gravistimulated conditions revealed few overlapping differentially expressed genes. Overexpression of each gene did not result in major branch angle differences. Shoot tip hormone levels were similar between tac1, lazy1, and Col, apart from exceptionally elevated levels of salicylic acid in lazy1. The data presented here provide a foundation for future study of TAC1 and LAZY1 regulation of shoot architecture.Lateral organ orientation in both shoots and roots plays a key role in a plant's interaction with the environment and its ability to access resources such as light and water. The IGT gene family members TILLER ANGLE CONTROL 1 (TAC1) and the related set of LAZY genes are important regulators of lateral organ orientation 1,2 . These genes share four conserved amino acid regions or domains, and LAZY genes share an additional C-terminal domain 3,4 . TAC1 generally occurs as a single copy gene, but many species possess multiple LAZY genes, with six identified in Arabidopsis 2-8 . While the LAZY1 gene of Arabidopsis thaliana (Arabidopsis) contributes almost exclusively to shoot architecture, the remaining Arabidopsis LAZY genes primarily control root architecture 7,8 . However, GUS reporter activity and additive shoot phenotypes in plants with combinatorial lazy1, lazy2, and lazy4 mutations suggest LAZY2 and LAZY4 also have a role in regulating shoot orientation 7,8 . LAZY2, 3, and 4 are also known as DEEPER ROOTING (DRO3, 2, & 1) and NEGATIVE GRAVITROPIC RESPONSE OF ROOTS (NGR1, 3, & 2), respectively, as they were separately identified as regulators of lateral root orientation 6,9-12 . Further, an alternate LAZY gene nomenclature denotes LAZY3 as LZY2 and LAZY4 as LZ3Y 8 .Lateral branches, tillers, leaves, and flower buds of plants with loss-of-function tac1 mutations or reduced TAC1 expression exhibit upright orientations 3,13-19 . In contrast, lazy1 mutants have wider branch or tiller angles and lazy4/dro1 mutants display prostrate lateral root orientations 4-9,20-24 . Plants with multiple lazy/dro mutations exhibit even wider lateral shoot/root...

IGT/LAZY family genes are differentially influenced by light signals and collectively required for light-induced changes to branch angle

“…In continuous dark, lazy1 branches began to grow upward, and displayed a phenotype similar to wild-type plants, suggesting that lazy1 has a light-conditional mutant phenotype. Loss of TAC1 did not appear to alter this re-orientation response, rather lazy1 appeared to be completely epistatic to tac1, which we reported under normal light conditions as well (Hollender et al, 2020). However, loss of DRO1 and DRO3 in addition to LAZY1 did lead to a loss of upward re-orientation in continuous darkness (Fig.…”

“…6). We observed an upward leaf curling phenotype, an overexpression phenotype described with other IGT genes (Guseman et al, 2017;Hollender et al, 2020), suggesting that the overexpression construct is at least partially functional. However, constitutive expression of LAZY1 under the 35S promoter did not fully rescue under continuous light or dark conditions (Fig.…”

“…The resulting LAZY6 CRISPR line shown here contains a single G insertion at nucleotide position 229 of the cDNA, resulting in a premature stop after the 80 th codon, and was phenotypically representative of multiple CRISPR mutants. The 35S::DRO1 and 35S::LAZY1 constructs were cloned as preciously described (Guseman et al, 2017;Hollender et al, 2020). Similarly to these, the 35S::LAZY6 construct was cloned by ligating the LAZY6 (At3g27025) CDS into the multiple cloning site downstream of the 35S promoter of a modified pBIN-AFRS expression vector (Belknap et al, 2008).…”

“…Unfortunately, after testing multiple primer pairs, we were unable to find efficient quantitative PCR primers that could consistently detect DRO3 (At1g17400) (data not shown). To minimize environmental variables, we measured expression responses for most light treatments using populations of 2 week-old seedlings grown on agar plates, in which LAZY and DRO gene expression has been determined (Yoshihara et al, 2013;Yoshihara and Spalding, 2017;Guseman et al, 2017;Hollender et al, 2020).…”

“…First, we grew Arabidopsis wild-type, lazy1 single, tac1 lazy1 double, and lazy1 dro1 dro3 triple mutants under continuous light and continuous dark regimes (Taniguchi et al, 2017;Yoshihara and Spalding, 2017;Hollender et al, 2020). Adult plants were grown in long-day conditions until they began initiating branches, and then were moved into continuous light or continuous dark.…”