Floral organ abscission peptide IDA and its HAE/HSL2 receptors control cell separation during lateral root emergence

Abstract

Throughout their life cycle, plants produce new organs, such as leaves, flowers, and lateral roots. Organs that have served their purpose may be shed after breakdown of primary cell walls between adjacent cell files at the site of detachment. In Arabidopsis, floral organs abscise after pollination, and this cell separation event is controlled by the peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which signals through the leucine-rich repeat receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2). Emergence of new lateral root primordia, initiated deep inside the root under the influence of auxin, is similarly dependent on cell wall dissolution between cells in the overlaying endodermal, cortical, and epidermal tissues. Here we show that this process requires IDA, HAE, and HSL2. Mutation in these genes constrains the passage of the growing lateral root primordia through the overlaying layers, resulting in altered shapes of the lateral root primordia and of the overlaying cells. The HAE and HSL2 receptors are redundant in function during floral organ abscission, but during lateral root emergence they are differentially involved in regulating cell wall remodeling genes. In the root, IDA is strongly auxin-inducible and dependent on key regulators of lateral root emergence--the auxin influx carrier LIKE AUX1-3 and AUXIN RESPONSE FACTOR7. The expression levels of the receptor genes are only transiently induced by auxin, suggesting they are limiting factors for cell separation. We conclude that elements of the same cell separation signaling module have been adapted to function in different developmental programs.

Fig. 1.

Mutations in IDA, HAE, and HSL2 delay LR emergence. (A) LR density (number of LRs per cm root) for ida (n = 21), hae hsl2 (n = 22), and WT plants (n = 20) 12 d after stratification. *Significant deviation from WT based on Student t test. (B) Schematic presentation of the stages of LR development. (C) Percentage of LRP at stages I and II for ida (n = 68), hae (n = 68), hsl2 (n = 81), hae hsl2 (n = 81), and WT plants (n = 69), 18 h after LR induction. (D) Percentage of LRP at stages IV to VIII (emerged) for ida (n = 68), hae (n = 59), hsl2 (n = 58), hae hsl2 (n = 56), and WT plants (n = 62), 42 h after LR induction. (E) Percentage of LRP with tips positioned in the overlaying EN, CO, and EP tissues or touching the CO or EP layer (tCO and tEP, respectively) for WT and mutants, as indicated. EM, emerged LRs. Numbers as in D. (F and G) WT and mutant LRP, as indicated, at stage V (F) and stage VI to VII (G). Overlaying EN, CO, and EP cells are indicated in orange, green, and blue arrowheads, respectively. (H) Percentage of stage VI LRP with tips positioned in the overlaying EN, CO, and EP tissues or touching the CO or EP layer (tCO and tEP, respectively) for WT and mutants, as indicated. (I) Percentage of stage VII LRP with tips positioned in the overlaying CO and EP tissues or touching the CO or EP layer (tCO and tEP, respectively) for WT and mutants, as indicated. (J) Cryo SEM images of emerged WT, hae, and ida LRs. The EP cells on each side of the LR have been colored. Arrows, symmetrical openings between EP cells in WT. Arrowheads, rifts in flattened and ruptured EP cells of mutant. (Scale bar: 50 μM.)

Fig. 2.

IDA, HAE, and HSL2 are expressed in cells overlaying new LRP and induced by auxin. (A) pIDA:GUS expression shown for the stages indicated. (B) Confocal images of pHSL2:YFP expression at stage III. (Left) PI-stained root; (Center) YFP signal; (Right) merge. The outline of the LRP is shown. Note that PI does not penetrate the EN or the LRP and therefore does not stain the inner EN wall and LR cell walls. (C and D) Confocal images of pHAE:HAE-YFP expression in hae hsl2 mutant (stage II) and WT background (stage IV), respectively. Note that the pHAE:HAE-YFP construct rescued the hae hsl2 mutants phenotypes (Fig. S5 A and B). Annotation as in B. (E–G) Relative expression levels of IDA, HAE, and HSL2 in roots in the presence of 1 μM IAA in WT, arf7, and arf19 background determined by RT-qPCR. The WT expression level before IAA addition was set to 1. SDs are shown. *Significant differences between WT and both mutants (P < 0.05).

Fig. 3.

IDA, HAE, and HSL2 influence cell wall remodeling and degradation. (A) 3D Z-stack confocal image of PI-stained WT root at the site of a stage I WT LR. The LRP has been colored yellow. (B) 2D confocal image showing scan (white graph) of the fluorescence from PI-stained EN, CO, and EP cell walls at the site of WT LRP (green arrowheads). Control scans were taken below and above the LRP (light green arrowhead). >, position of LRP. (C–F) Confocal images of PI-stained roots at the site of a stage I LRs for hsl2 (2D), ida (2D), and hae (3D, yellow colored LRP) and hae hsl2 (2D). (G) PI fluorescence of LRP-overlaying cell walls between EN and CO relative to the reference point at the opposite side based on scans for six to nine roots. Bars indicate SD. Asterisks denote significant differences from fluorescence at control positions relative to the same reference point (Ctr) (*P < 0.05; ***P < 0.001, Student t test). (H and I) Expression of pXTR6:GUS and pPGAZAT:GUS and in WT, ida, and hae hsl2 background at the LR stages indicated. (J) Expression levels of PGAZAT and PGLR in mutant 7-d-old roots relative to WT determined by RT-qPCR. Bars indicate SD. *Significant deviation from WT (P < 0.05). (K) Stage VII LRP of 35S:IDA and 35S:IDA hae hsl2, as indicated. Arrowheads, cell walls of overlaying CO and EP cells. (Scale bars: 30 μM in 3D images, 20 μM in 2D images.)

Fig. 4.

Model of IDA-HAE/HSL2 signaling in LR emergence. IDA expression and signaling to open a gateway for the LRP is hypothesized to occur in a wave-like fashion in the cells directly overlaying the developing LRPs. IDA is suggested induced in the EN by auxin diffusing from the young LRP and secreted to interact with HAE and HSL2 receptors already present, to trigger expression of CWR genes, leading to loosening and breakdown of the cell wall of the EN cells directly overlaying LRPs. Expression of IDA in the CO and EP overlaying the LRP is dependent on auxin influx via LAX3 and the auxin-dependent cascade leading to activation of ARF7, which also transiently induces expression of the receptor genes. HAE and HSL2 function redundantly to up-regulate some CWR genes like XTR6, for instance at the base of the LRP, whereas IDA signaling, mainly through the HAE receptor, in overlaying tissues will induce downstream genes encoding cell wall degradation enzymes like PGs, leading to cell separation.