Directional organ growth allows the plant root system to strategically cover its surroundings. Intercellular auxin transport is aligned with the gravity vector in the primary root tips, facilitating downward organ bending at the lower root flank. Here we show that cytokinin signaling functions as a lateral root specific anti-gravitropic component, promoting the radial distribution of the root system. We performed a genome-wide association study and reveal that signal peptide processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, thereby, determines the degradation of cytokinins in natural Arabidopsis thaliana accessions. Cytokinin signaling interferes with growth at the upper lateral root flank and thereby prevents downward bending. Our interdisciplinary approach proposes that two phytohormonal cues at opposite organ flanks counterbalance each other’s negative impact on growth, suppressing organ growth towards gravity and allow for radial expansion of the root system.

Commercially available fluorescent dyes enable the fast and specific visualization of plant vacuoles, allowing for investigation of membrane dynamics and vacuolar biogenesis in living cells. Here, we describe different approaches tinting the tonoplast or the vacuolar lumen with a range of dyes, and illustrate its utilization with established fluorescent-tagged marker lines.

HighlightPIN2 shows distinct trafficking and vacuolar turnover in neighbouring tricho- and atrichoblast cell files. Differential abundance of PIN2 in the root epidermis could have developmental importance for root gravitropism.

The vacuole has a space-filling function, allowing a particularly rapid plant cell expansion with very little increase in cytosolic content (Löfke et al., 2015; Scheuring et al., 2016; Dünser et al., 2019). Despite its importance for cell size determination in plants, very little is known about the mechanisms that define vacuolar size. Here, we show that the cellular and vacuolar size expansions are coordinated. By developing a pharmacological tool, we enabled the investigation of membrane delivery to the vacuole during cellular expansion. Our data reveal that endocytic membrane sorting from the plasma membrane to the vacuole is enhanced in the course of rapid root cell expansion. While this ‘compromise’ mechanism may theoretically at first decelerate cell surface enlargements, it fuels vacuolar expansion and, thereby, ensures the coordinated augmentation of vacuolar occupancy in dynamically expanding plant cells.

31Directional organ growth allows the plant root system to strategically cover its 32 surroundings. Intercellular auxin transport is aligned with the gravity vector in 33 the primary root tips, facilitating downward organ bending at the lower root 34 flank. Here we show that cytokinin signaling functions as a lateral root specific 35 anti-gravitropic component, promoting the radial distribution of the root system. 36 We performed a genome-wide association study and revealed that signal peptide 37 processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, 38 thereby, determines the degradation of cytokinins in natural Arabidopsis 39 thaliana accessions. Cytokinin signaling interferes with growth at the upper 40 lateral root flank and thereby prevents downward bending. Our interdisciplinary 41 approach revealed that two phytohormonal cues at opposite organ flanks 42 counterbalance each other's negative impact on growth, suppressing organ 43 growth towards gravity and allow for radial expansion of the root system. 44 45 128 regulate angular growth of LRs, we initially transferred 7-day old seedlings of the 129 reference accession Col-0 to medium supplemented with CKs. We observed a 130 concentration-dependent increase in GSA values of LRs emerging in presence of 131 6active CKs, such as 6-Benzylaminopurin (BAP) ( Figure 2D), trans-zeatin (tZ) and 132 isopentenyladenine (iP) ( Figure S3F and G). Conversely, CK receptor mutants showed 133 accelerated bending of LRs and accordingly decreased GSA values ( Figure 2E). 134These data suggest that cytokinin signaling interferes with downward bending of 135 emerged LRs. Notably, emerging LRs of winter oilseed rape also displayed reduced 136 bending of LRs when treated with BAP ( Figure S3H), suggesting that the effect of CK 137 on directional LR growth is likely to be conserved. 138To further assess the importance of CKX2 in GSA establishment, we disrupted 139 CKX activity in the reference accession Col-0. Treatments with the CKX inhibitor 140 INCYDE (Zatloukal et al., 2008) phenocopied the ckx2 loss-of-function mutant, both 141 displaying more horizontal LRs when compared to its respective controls (Figure 2F, 142 G). On the other hand, CKX2 overexpressing (OX) plants showed accelerated bending 143 of LRs, phenocopying the CK receptor mutants (Figure 2G). These data suggest that 144 CK signaling defines directional lateral root growth by reducing LR bending after 145 emergence. 146 147 157 ARR4 (pARR3:GUS/ pARR4:GUS; Figure S4C)). Moreover, angular growth of LRs 158 was not altered in arr3 or arr4 mutants ( Figure S4D). On the other hand, we confirmed 159 expression of CRF2 and CRF3 in the early stages of LR development ( Figure 3A and 160 S4E). pCRF2:GFP-GUS was ubiquitously expressed in young LRs, while pCRF3:GFP-161 GUS was preferentially expressed in cortical and epidermal cell files ( Figure 3A and 162 S4E). Notably, compared to emerged laterals, the main root displayed much weaker 163 CRF2 and CRF3 expression ( Figure S4F), suggesting that CRF2 an...