The HOS1-PIF4/5 module controls callus formation in Arabidopsis leaf explants

ABSTRACT A two-step plant regeneration has been widely exploited to genetic manipulation and genome engineering in plants. Despite technical importance, understanding of molecular mechanism underlying in vitro plant regeneration remains to be fully elucidated. Here, we found that the HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1)-PHYTOCHROME INTERACTING FACTOR 4/5 (PIF4/5) module participates in callus formation. Consistent with the repressive role of HOS1 in PIF transcriptional activation activity, hos1–3 mutant leaf explants exhibited enhanced callus formation, whereas pif4–101 pif5–3 mutant leaf explants showed reduced callus size. The HOS1-PIF4/5 function would be largely dependent on auxin biosynthesis and signaling, which are essential for callus initiation and proliferation. Our findings suggest that the HOS1-PIF4/5 module plays a pivotal role in auxin-dependent callus formation in Arabidopsis.

Some plant cells can form a mass of pluripotent cells, called callus.This not only occurs spontaneously at wound sites but can also be induced by in vitro tissue cultures.2][3] During in vitro tissue culture, a concentration ratio of two phytohormones, auxin and cytokinin, determines cell fate transitions: incubation of tissue explants on auxin-rich callus-inducing medium (CIM) facilitates callus proliferation, 3,4 whereas de novo shoot regeneration can be stimulated by incubation on the cytokininrich shoot-inducing medium (SIM). 3 particular emphasis has been placed on the callus formation process, because active callus proliferation facilitates pluripotency acquisition, which is a fundamental basis of plant regeneration. 5Accumulating evidence has shown that the CIM-derived callus resembles root primordium, regardless of origin of tissue explants. 6In Arabidopsis, callus formation is initiated from the pericyclelike cells, 4,7 which then undergo asymmetric cell division to establish root primordium identity with the activation of auxin-inducible root developmental genes, such as AUXIN RESPONSE FACTORs (ARFs) and LATERAL ORGAN BOUNDARIES DOMAINs (LBDs). 8After acquisition of root primordium characteristics, callus cells establish a regeneration competence via expression of root stem cell regulators, including PLETHORAs (PLTs), SCARECROW (SCR), WUSCHEL-RELATED HOMEOBOX 5 (WOX5), WOX7, and WOX14. 6,7,9Accordingly, plt3 plt5 plt7, scr, and wox5 wox7 wox14 mutants exhibit impaired de novo shoot regeneration due to the failure of pluripotency acquisition. 9,102][13][14][15] In particular, the HOS1 protein is important for controlling auxin biosynthesis and signaling.The hos1 mutants exhibited elongated hypocotyls possibly with increased auxin biosynthesis compared to wild-type in light condition. 12In addition, HOS1 also inhibits the transcriptional activation activity of PIF4 to further influence auxin signaling-related genes. 15lthough plant tissue culture has been widely exploited to genome engineering of various plant species, its application is still limited in many plant species.7][18] To find out genetic obstacles for efficient plant regeneration, we screened several genetic mutants and tried to find mutants displaying enhanced callus formation and/or shoot regeneration.Our initial screening suggested that a hos1 mutant showed an enhanced callus formation compared to wild type.To validate our preliminary results, we obtained two HOS1-deficient mutant alleles and examined callus formation rate.As expected, callus formation was significantly increased in hos1 mutants compared to wild type, especially at a low concentration of exogenous auxin in CIM (Figure 1a).
We next asked how HOS1 regulates callus formation.Since auxin signaling is closely associated with callus formation, [19][20][21] we suspected that HOS1 might affect auxin biosynthesis and/or signaling.Interestingly, HOS1 is known to inhibit the transcriptional activation activity of PIF4, key regulator of auxin biosynthesis and signaling, without affecting its transcript accumulation. 15onsistent with this, an auxin-responsive gene regulated by PIF4/5 was upregulated in hos1 mutants (Supplemental Figure S1).We thus examined whether PIF4 and its redundant gene PIF5 are indeed involved in callus formation.The phenotypic analysis showed that the pif4-101 pif5-3 mutant exhibited smaller callus size than wild type (Figure 1b).To confirm the genetic hierarchy, we checked callus proliferation activity of hos1-3 pif4-101 pif5-3 triple mutant.As a result, the enhanced callus size phenotype of hos1-3 leaf explants was compromised in part by introducing pif4-101 pif5-3 mutations (Figure 1c).These results indicate that the HOS1-PIF4/5 module plays an important role in regulating callus formation in Arabidopsis.Although HOS1 is likely dependent on PIF4 and PIF5 in the control of callus proliferation, we cannot rule out the possibility that additional biological functions of HOS1, such as ethylene signaling and circadian control, 14,22 could also be linked to plant regeneration. 23,24urthermore, considering the enhanced greening phenotype in pif4-101 pif5-3 mutant leaf explants (Figure 1b), there might be additional developmental impact of the HOS1-PIF4/5 module in leaf senescence and/or cytokinin signaling control, which can influence in vitro plant regeneration.
Given that the HOS1 protein accumulated during callus proliferation (Figure 1d), this protein might constitute a negative feedback pathway of auxin biosynthesis and/or signaling that allows proper callus division rate during in vitro tissue culture.In this context, HOS1 may act as a genetic barrier that limits callus initiation as well as callus proliferation by restricting auxin biosynthesis and signaling.Since HOS1 is widely conserved across various plant species, 25 inactivation of HOS1 may contribute to enhancing callus formation especially in crop and woody plants that have a low capability of callus formation.

Immunoblot analysis
Harvested plant materials were ground in liquid nitrogen, and total cellular extracts were suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer.Protein samples were analyzed using SDS-PAGE (10% gels) and blotted onto Hybond-P+ membranes (Amersham-Pharmacia). Proteins were immunologically detected using anti-GFP antibody (Abcam, ab290).

Figure 1 .
Figure 1.The HOS1-PIF4/5 module regulates callus formation.(a) Role of HOS1 in callus formation.(b) Reduced callus formation in pif4-101 pif5-3 mutant leaf explants.In (a) and (b), the third and fourth leaves of 2-week-old seedlings were used to induce calli on callus-inducing medium (CIM) supplemented with different concentrations of 2,4-D.Plates were incubated for 2 weeks under continuous dark conditions and photographed (left panels).Thirty calli excised from leaf explants were collected to measure fresh weight (right panels).Data represent the mean ± SEM.Asterisks indicate statistically significant differences (Student's t-test, *P < 0.05; ***P < 0.001).Bars indicate the standard error of the mean.Scale bars = 5 mm.(c) Genetic analysis.Leaf explants from the third and fourth leaves of 2-week-old seedlings were used to induce calli on CIM supplemented with 0.1 µg/mL 2,4-D.Plates were incubated for 2 weeks under continuous dark conditions.Thirty calli excised from leaf explants were collected to measure fresh weight.Data represent the mean ± SEM.Statistically significant differences were determined using one-way analysis of variance (ANOVA), followed by Fisher's post hoc test.Different letters indicate significant differences (*P < 0.05).Scale bars = 5 mm.(d) Accumulation of HOS1 during callus formation.Leaf explants from the third and fourth leaves of 2-week-old seedlings were used to induce calli on CIM.Leaf explants of pHOS1:HOS1-eGFP were incubated on CIM supplemented with 0.1 µg/mL 2,4-D for up to 7 d.Whole leaf explants with developing calli were collected for immunoblot analysis.The HOS1-eGFP protein was detected immunologically using an anti-GFP antibody.Coomassie blue-stained gel is shown as a loading control.DAC, days after incubation on CIM.