() Loss of Function Mutations in the Arabidopsis Heterotrimeric G protein a Subunit Enhance the Developmental Defects of Brassinosteroid Signaling and Biosynthesis Mutants Yajun Gao 1, 2, Shucai Wang[.]
Plant Cell Physiol 49(7): 1013–1024 (2008) doi:10.1093/pcp/pcn078, available online at www.pcp.oxfordjournals.org ß The Author 2008 Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists All rights reserved For permissions, please email: journals.permissions@oxfordjournals.org Loss-of-Function Mutations in the Arabidopsis Heterotrimeric G-protein a Subunit Enhance the Developmental Defects of Brassinosteroid Signaling and Biosynthesis Mutants Yajun Gao 1, , Shucai Wang 1, Tadao Asami and Jin-Gui Chen 1, * Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada College of Resources and Environment, Northwest Agriculture and Forestry University, Yangling, Shaanxi 712100, PR China Plant Science Center and Plant Functions Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan Loss-of-function alleles of the sole heterotrimeric G-protein a subunit in Arabidopsis, GPA1, display defects in cell proliferation throughout plant development Previous studies indicated that GPA1 is involved in brassinosteroid (BR) response Here we provide genetic evidence that lossof-function mutations in GPA1, gpa1-2 and gpa1-4, enhance the developmental defects of bri1-5, a weak allele of a BR receptor mutant, and det2-1, a BR-deficient mutant in Arabidopsis gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants had shorter hypocotyls, shorter roots and fewer lateral roots, and displayed more severe dwarfism than bri1-5 and det2-1 single mutants, respectively By using the Arabidopsis hypocotyl as a model system where the parameters of cell division and cell elongation can be simultaneously measured, we found that gpa1 can specifically enhance the cell division defects of bri1-5 and det2-1 mutants Similarly, gpa1 specifically enhances cell division defects in the primary roots of bri1-5 and det2-1 mutants Furthermore, an additive effect on cell division between gpa1 and bri1-5 or det2-1 mutations was observed in the hypocotyls, whereas a synergistic effect was observed in the roots Taken together, these results provided the first genetic evidence that G-protein- and BR-mediated pathways may be converged to modulate cell proliferation in a cell/tissue-specific manner Keywords: Arabidopsis — Brassinosteroid (BR) — Cell proliferation — Heterotrimeric G-protein a subunit (GPA1) — Hypocotyl — Root Abbreviations: BR, brassinosteroid; GPA1, Arabidopsis heterotrimeric G-protein a subunit; GPCR, G-protein-coupled receptor; G-proteins, heterotrimeric guanine nucleotide-binding proteins Introduction The heterotrimeric guanine nucleotide-binding proteins (G-proteins) act as critical molecular switches in diverse signal transduction pathways in eukaryotes (Gilman 1987, Hamm 1998, Neubig and Siderovski 2002, Pierce et al 2002) Plants use G-proteins to regulate multiple developmental processes and hormone responses (reviewed by Perfus-Barbeoch et al 2004, Chen 2008) Loss-of-function mutations in the G-protein a subunit (Ga) and b subunit (Gb) in plants confer altered sensitivities to multiple hormones including auxin, ABA, gibberellins, brassinosteroids (BRs) and jasmonic acid (Ueguchi-Tanaka et al 2000, Wang et al 2001, Ullah et al 2002, Ullah et al 2003, Chen et al 2004, Trusov et al 2006, Wang et al 2006, Trusov et al 2007) Many morphological phenotypes observed in the loss-of-function mutants of Arabidopsis Ga and Gb subunits are attributed to the modulatory role of G-proteins in cell proliferation (Ullah et al 2001, Chen et al 2003, Ullah et al 2003, Chen et al 2006) G-proteins couple the recognition of extracellular signals by cell surface G-protein-coupled receptors (GPCRs) to activation of downstream effectors (Gilman 1987) In the classical signaling paradigm, upon GPCR activation, the Ga of the G-protein complex undergoes a conformational change, resulting in GDP/GTP exchange and the dissociation of Gbg dimer from the complex Activated Ga (GTP-bound) and liberated Gbg then bind to downstream effectors In Arabidopsis, the Ga is encoded by a single gene, GPA1 (Ma et al 1990) Although GPA1 plays a regulatory role in multiple developmental processes and hormonal responses, the upstream (GPCR) and downstream (effector) components in the G-protein signaling pathway remain largely elusive GPCRs are proteins that typically have seven-transmembrane domains There are several dozen proteins that contain seven-transmembrane domains in Arabidopsis (Moriyama et al 2006, Temple and Jones 2007) However, only two such proteins, GCR1 and AtRGS1, have been shown to bind GPA1 physically (Chen et al 2003, Pandey and Assmann 2004), but no ligand has been identified for either GCR1 or AtRGS1 Recently, GCR2 has been proposed to be a GPCR for ABA (Liu et al 2007) However, GCR2 was not clearly predicted as a seven-transmembrane domain-containing protein (Gao et al 2007, Johnston et al 2007, Illingworth et al 2008) *Corresponding author: E-mail, jingui@interchange.ubc.ca; Fax, ỵ1-604-822-6089 1013 Heterotrimeric G-proteins and brassinosteroid Results Loss-of-function mutations in GPA1 enhance the dwarfism of BR receptor and biosynthesis mutants The Ga subunit of the heterotrimeric G-protein complex is encoded by a single gene, GPA1, in Arabidopsis Previous studies indicated that the GPA1 may be involved in BR responses (Ullah et al 2002, Chen et al 2004) In order to obtain insight into the relationship between GPA1- and the BR-mediated pathways, we generated double mutants between gpa1-2 (Ullah et al 2001), a lossof-function allele of GPA1, and bri1-5 (Li and Chory 1997, Noguchi et al 1999a), a weak mutant allele of BRI1 which encodes a receptor for BR Both gpa1-2 and bri1-5 are in the Wassilewskija (WS) ecotypic background Because the regulation of BR homeostasis is an important step in BR signaling and because both BR receptor and biosynthesis mutants are dwarf, we also generated double mutants between gpa1-4, a loss-of-function allele of GPA1 in the Columbia (Col) ecotypic background, and det2-1 (in the Col ecotypic background), a mutant which has a defect in the A B Plant height (cm) 15 12 * # * gp WS a1 -2 gp a1 bri1 -2 br i1 -5 5 br i1- br i1- gp a1 -2 -2 gp a1 W S C Plant height (cm) D 15 12 * # * gp Co a1 l a1 det -4 2de t2 -1 gp -1 gp a1 -4 de t2 de t2 -1 gp a1 -4 ol There are several proteins that could act as potential downstream effectors for GPA1, including AtPirin1 (Lapik and Kaufman 2003), PLDa1 (Zhao and Wang 2004), PD1 (Warpeha et al 2006) and THF1 (Huang et al 2006) However, a complete cascade in any given developmental process or hormonal response mediated by the G-proteins is still lacking, and the mechanism through which G-proteins regulate phenotypic and developmental plasticity remains unclear (Assmann 2004) Previous studies indicated that a loss-of-function allele of the Ga subunit in Arabidopsis, GPA1, displayed reduced sensitivities to brassinolide, a biologically active form of BR, in hypocotyl growth elongation and seed germination assays (Ullah et al 2002) In rice, a loss-of-function allele of Ga, d1, displayed slightly reduced sensitivity to BR in BR-induced OsBLE2 expression (Yang et al 2003) In the assays of root elongation inhibition, lamina inclination promotion and coleoptile elongation promotion, rice d1 mutants also displayed reduced sensitivities to BR (Wang et al 2006) These findings suggested that G-proteins may have a role in the BR-mediated pathway, which led us to investigate further the relationship between the G-proteins and the key components of BR signaling pathways In particular, we wanted to investigate the relationship between GPA1, the sole Ga subunit in Arabidopsis, and BRI1, a receptor for BR, in the modulation of cell proliferation We demonstrate that the loss-of-function mutations in GPA1 specifically enhance the cell division defects in bri1-5, a weak mutant allele of BRI1 We propose that GPA1- and BR-mediated pathways may act in concert to modulate cell proliferation C 1014 Fig Plant heights of gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants (A) Phenotype of a 58-day-old gpa1-2 bri1-5 double mutant (B) Height of a gpa1-2 bri1-5 double mutant (C) Phenotype of a 58-day-old gpa1-4 det2-1 double mutant (D) Height of a gpa1-4 det2-1 double mutant Wild-type and mutant plants were grown under identical conditions with a 14/10 h photoperiod Scale bars in (A) and (C), cm The heights of plants were measured at maturity Shown are the average heights of at least 10 plants for each genotype SE significant difference from the wild-type (WS or Col), P50.05 #significant difference from bri1-5 or det2-1, P50.05 early step of BR biosynthesis (Li et al 1996, Fujioka et al 1997, Li et al 1997, Noguchi et al 1999b) Under normal growth conditions (e.g 14/10 h photoperiod at 140 mmol m–2 s–1, 238C), gpa1 mutants not display dwarfism whereas bri1-5 and det2-1 are dwarf, consistent with previous reports (Li et al 1996, Fujioka et al 1997, Li and Chory 1997, Li et al 1997, Noguchi et al 1999a, Noguchi et al 1999b, Ullah et al 2001, Ullah et al 2003) Interestingly, we found that gpa1 mutations could significantly enhance the dwarfism of bri1-5 and det2-1 mutants (Fig 1), suggestive of a possible genetic interaction between GPA1- and BR-mediated pathways Heterotrimeric G-proteins and brassinosteroid BR receptor mutants and biosynthesis mutants have defects in both cell elongation and cell division in hypocotyls Because BR has a prominent role in regulating cell elongation whereas GPA1 is a critical modulator for cell division, we wanted to investigate further the cellular basis of the observed enhanced phenotypes of bri1-5 and det2-1 by gpa1 mutations (Fig 1) However, the plant height reflects the combined effect of cell division at the shoot apical meristem and at the intercalary meristem and cell elongation along the inflorescence stem In addition, the cell division and cell elongation in the primary inflorescence stem vary at different developmental stages bri1-5 and det2-1 mutants are slightly late flowering, whereas gpa1 mutants have wild-type flowering time These factors made a direct measurement and comparison of cell division and cell elongation on the inflorescence stems of these single and double mutants unreliable Therefore, we sought a reliable and robust system in which cell division and cell elongation can be simultaneously measured and compared in these mutants For this purpose, we selected the hypocotyls, because the number of cells in the epidermis and cortex is pre-determined during embryogenesis and little cell division occurs in the epidermis or cortex in dark- or light-grown Arabidopsis seedlings (Gendreau et al 1997) A shorter hypocotyl, compared with the wild type, in any given mutants could be due to a defect in either cell division (pre-determined during embryogenesis) or cell elongation (determined post-embryogenesis), or both By counting the number of cells and measuring the length of cells in a single cell file longitudinally from the base to the top of a hypocotyl, defects in cell division and/or cell elongation can be simultaneously determined More importantly, this approach had been used previously to determine the cellular basis of the short hypocotyl phenotype of gpa1 mutants (Ullah et al 2001), and a loss-of-function allele of the sole Arabidopsis Gb (Ullah et al 2003) As reported previously (Ullah et al 2001, Chen et al., 2003, Jones et al 2003, Chen et al 2004, Chen et al 2006), etiolated seedlings of the gpa1 mutants, gpa1-2 and gpa1-4, have short hypocotyls and partially opened hooks (Fig 2A–D) The phenotype of gpa1-2 is comparable with that of bri1-5, but bri1-5 mutants are about 30% shorter than gpa1 mutants (Fig 2A, B) The phenotype of gpa1-4 is similar to that of det2-1, but det2-1 mutants have even shorter hypocotyl and are hookless (Fig 2C, D) Consistent with these observations, gpa1-2 and gpa1-4 mutants are also similar to seedlings treated with Brz2001, a specific BR biosynthesis inhibitor (Sekimata et al 2001) On the basis of these results, we concluded that both GPA1 and BR have a role in regulating hypocotyl growth and that the Arabidopsis hypocotyl is a suitable system for dissecting the role of GPA1 and BR in regulating cell elongation and cell division 1015 Although it has been recognized for many years that etiolated bri1-5 and det2-1 mutant seedlings have shorter hypocotyls, compared with the wild type, the cellular basis for such shortness was unclear Because BR has a prominent role in regulating cell elongation over cell division, we expected that the reduced lengths of hypocotyls in bri1-5 and det2-1 mutants were due to the defect in cell elongation Indeed, we found that the length of hypocotyl epidermal cells was dramatically reduced in bri1-5 and det2-1 mutants (Fig 2E, F) Interestingly, we found that the number of epidermal cells in bri1-5 and det2-1 mutants was also significantly reduced (Fig 2E, F) From the base (hypocotyl/root junction) to the top (lateral to cotyledons) of a hypocotyl, wild-type (WS or Col) plants have about 21 cells in a single cell file longitudinally However, bri1-5 and det2-1 mutants had only about 15 cells (Fig 2E, F) These results provided direct evidence that BR has a role in regulating hypocotyl cell division and that the short hypocotyls of bri1-5 and det2-1 mutants are due to reductions in both cell elongation and cell division We extended our analysis to other cell types in the hypocotyl Arabidopsis hypocotyls have two layers of cortex cells, outer and inner cortex cells We counted the number and measured the length of outer cortex cells (located just beneath the epidermal cells) in a single cell file longitudinally from the base to the top of a hypocotyl We found that the length of outer cortex cells was dramatically reduced in the hypocotyls of bri1-5 and det2-1 mutants, compared with that in wild-type (Supplementary Fig S1) Similar to that of epidermal cells, the number of outer cortex cells was also significantly reduced in the hypocotyls of bri1-5 and det2-1 mutants (Supplementary Fig S1) These results supported the view that the short hypocotyls of bri1-5 and det2-1 mutants are due to reductions in both cell elongation and cell division Because epidermal cells on the plant surface are generally believed to have a more important role than cortex cells in determining the shape of an organ, and epidermal cells on hypocotyls can be easily recognized due to their location and shape, in our subsequent experiments we specifically focused on the epidermal cells to study cell division and cell elongation in hypocotyls We compared the cell number (indicator of cell division) and cell length (indicator of cell elongation) in gpa1, bri1-5 and det2-1 single and double mutants to determine the relationship between GPA1 and BRI or DET2 in regulating cell division and cell elongation in hypocotyls Because the cell division defect in the hypocotyls of BR signaling or biosynthesis mutants had not been reported previously and because bri1-5 is a weak mutant allele of BRI1, we wanted to examine further if such a defect also occurs in a strong mutant allele of BRI1 Therefore, we used the same assay to examine the number and length of Heterotrimeric G-proteins and brassinosteroid E Epidermal cell length (mm) l gp a1 Co W S C -4 de t2 -1 Co l+ B Br z2 + -2 i1 br gp a1 W S A rz 20 00 01 1016 * 4.0 * 2.0 6.0 5.0 4.0 * * 3.0 * 2.0 1.0 + bri1 Br -5 z2 00 S W C gp ol a1 Co -4 l + det Br 2-1 z2 00 S W a1gp 600 WS bri1-5 500 400 300 200 100 F Epidermal cell length (mm) * D Hypocotyl length (mm) Hypocotyl length (mm) 8.0 6.0 700 10 13 16 19 21 (Base) Cell number (Top) mm B 800 1000 800 Col det2-1 600 400 200 (Base) 10 13 16 19 21 Cell number (Top) Fig gpa1, bri1-5 and det2-1 mutants share the similar short hypocotyl phenotype in etiolated seedlings (A) Phenotype of 3-day-old, dark-grown mutant seedlings in the Wassilewskija (WS) ecotypic background (B) The hypocotyl length of 3-day-old, dark-grown mutant seedlings (C) Phenotype of 3-day-old, dark-grown mutant seedlings in the Columbia-0 (Col) ecotypic background (D) The hypocotyl length of 3-day-old, dark-grown mutant seedlings Brz2001, a specific biosynthesis inhibitor of BR, was applied at mM Scale bars in (A) and (C), mm significant difference from WS or Col, P50.05 Shown in (B) and (D) are the average lengths of hypocotyls from at least 20 seedlings for each genotype SE (E) The number and length of hypocotyl epidermal cells in 3-day-old, dark-grown WS and bri1-5 mutant seedlings (F) The number and length of hypocotyl epidermal cells in 3-day-old, dark-grown Col and det2-1 mutant seedlings Shown in (E) and (F) are the average cell lengths of 10 seedlings for each genotype SE hypocotyl epidermal cells in bri1-4 (Li and Chory 1997 Noguchi et al 1999a), a strong and null allele for BRI1 in the WS ecotypic background Because bri1-4 mutants rarely produce seeds, we picked up bri1-4 mutants from the progeny of a plant heterozygous for the bri1-4 locus In 4-day-old, light-grown seedlings, bri1-4 mutants, evident by their extreme shortness of hypocotyl and dark green cotyledons, were readily picked up from this segregating population We found that similar to that of the bri1-5 mutant, the short hypocotyl of bri1-4 was due to reductions in both cell elongation and cell division (Supplementary Fig S2) We also examined the cell number and length of hypocotyl epidermal cells in another BR-deficient mutant, dwf4-102 (Azpiroz et al 1998, Choe et al 1998, Nakamoto et al 2006), which is a T-DNA insertion mutant in the Col ecotypic background and has defects in the key steps of BR biosynthesis (Choe et al 1998) Again, we found that the short hypocotyl of dwf4-102 was due to reductions in both cell elongation and cell division (Supplementary Fig S2) Therefore, we concluded that BR regulates both cell elongation and cell division in hypocotyls Loss-of-function mutations in GPA1 enhance the cell division defect in the hypocotyls of bri1-5 and det2-1 mutants Having determined that both GPA1 and BR have a role in regulating cell division in the hypocotyl epidermal cells, we wanted to investigate the relationship between GPA1 and BR in the modulation of cell proliferation by examining cell elongation and cell division defects in gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants and comparing them with those in single mutants We found that the hypocotyls of gpa1-2 bri1-5 double mutants were significantly shorter than those of the bri1-5 single mutant when grown in the dark (Fig 3A, B) Similarly, in etiolated seedlings, the hypocotyls of gpa1-4 det2-1 double mutants 8.0 * * # * 2.0 -4 -1 8.0 * 6.0 4.0 * 2.0 # * ol C de t2 -1 de t2 -1 -4 a1 gp S W a1- i1-5 i1-5 r r gp b b a gp a1 4.0 D l gp 6.0 Co t2 a1 gp gp a1 -2 -5 i1 br gp C Hypocotyl length (mm) Hypocotyl length (mm) B a1 S -5 de -2 W br gp a1 -4 i1 A de t2 -1 Heterotrimeric G-proteins and brassinosteroid Fig Phenotype of dark-grown seedlings of gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants (A) Phenotype of 3-day-old, dark-grown gpa1-2 bri1-5 double mutants Scale bar, mm (B) Hypocotyl length of gpa1-2 bri1-5 double mutants significant difference from WS, P50.05 #significant difference from the bri1-5 mutant, P50.05 (C) Phenotype of 3-day-old, dark-grown gpa1-4 det2-1 double mutants Scale bar, mm (D) Hypocotyl length of gpa1-4 det2-1 double mutants significant difference from Col, P50.05 #significant difference from det2-1 mutant, P50.05 Shown in (B) and (D) are average lengths of hypocotyls of 20 seedlings for each genotype SE were significantly shorter than those of the det2-1 single mutant (Fig 3C, D) Light plays an important role in regulating hypocotyl growth The growth characteristics of hypocotyl epidermal cells in light-grown Arabidopsis seedlings are different from those in etiolated seedlings (Gendreau et al 1997) Therefore, we also measured the hypocotyl lengths of lightgrown gpa1-2 bri1-5 and gpa1-4 det2-1 mutant seedlings and compared them with those of bri1-5 and det2-1 single mutants, respectively Because bri1-5 and det2-1 mutants have very short hypocotyls under normal light conditions (e.g 140 mmol m–2 s–1), we adopted low-light conditions (30 mmol m–2 s–1) under which the hypocotyls of bri1-5 and det2-1 mutants are longer, making it easier and more accurate to quantify the number and length of epidermal cells 1017 We found that in low-light-grown seedlings, the hypocotyl lengths of bri1-5 and det2-1 were also further reduced by gpa1-2 and gpa1-4 mutations, respectively (Figs 4A, B, and 5A, B) Taken together, these results suggested that gpa1 mutations can enhance the hypocotyl phenotype of dark- or low-light-grown bri1-5 and det2-1 seedlings Subsequently, we counted the number and measured the length of hypocotyl epidermal cells in a single cell file longitudinally in gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants, and compared them with those in bri1-5 and det2-1 single mutants, respectively Interestingly, we found that the number of epidermal cells was further reduced in gpa1-2 bri1-5 double mutants, compared with gpa1-2 or bri1-5 single mutants (Fig 4C, F) gpa1-2 bri1-5 double mutants only had about 11 epidermal cells, compared with approximately 15 cells in gpa1-2 or bri1-5 single mutants and 21 cells in wild-type plants (Fig 4C, F) However, the cell elongation in the gpa1-2 bri1-5 double mutant, measured by the average length or maximal length of epidermal cells (Fig 4D–F), was similar to that in the bri1-5 single mutant Similarly, we found that that the number of epidermal cells in gpa1-4 det2-1 double mutants was significantly reduced, compared with that in gpa1-4 and det2-1 single mutants (Fig 5C, F), whereas the average length and maximal length of hypocotyl epidermal cells in gpa1-4 det2-1 double mutants were similar to those in det2-1 single mutants (Fig 5D–F) Taken together, these results suggested that gpa1 mutations can specifically enhance the cell division defects in the hypocotyls of bri1-5 and det2-1 mutants Loss-of-function mutations in GPA1 enhance the defect in root development of bri1-5 and det2-1 mutants We extended our analysis of the relationship between gpa1-2 and bri1-5, and between gpa1-4 and det2-1, to nonaerial organs, specifically the roots We measured the length of the primary root and counted the number of lateral roots to assess the impact on and relationship of these mutations to root development gpa1 mutants have primary roots of normal length and produced fewer lateral roots (Ullah et al 2003, Chen et al 2006), whereas both bri1-5 and det2-1 mutants have short primary roots and produced fewer lateral roots (Fig 6A, B, D, E) We found that gpa1 mutations significantly enhanced the short primary root phenotype of the bri1-5 and det2-1 mutants (Fig 6A, B, D, E) The number of lateral roots was also significantly reduced in gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants, compared with bri1-5 and det2-1 single mutants, respectively (Fig 6A, C, D, F) These results suggested that similar to the situation of aerial organs, gpa1 mutations can also enhance the defects in root development of bri1-5 and det2-1 mutants Heterotrimeric G-proteins and brassinosteroid 300 200 100 10 13 Cell number 16 19 21 (Top) gp Co a l gp a1 de 1-4 -4 t de -1 t2 -1 -1 de t2 -1 250 200 150 100 * * 50 600 500 400 300 200 * * 100 0 gp Co a l a1 de 1-4 -4 t2 de -1 t2 -1 F 300 600 gp Co a l a1 d 1-4 -4 et2 de -1 t2 -1 E 350 gp # * 10 gp bri1-5 gpa1-2 bri1-5 ** 15 gp gp WS a1 a1 br -2 -2 i1br i1 -5 400 Maximal cell length (mm) 20 500 # * * 1.0 gp Co a l a1 de 1-4 -4 t2 de -1 t2 -1 100 gpa1-2 2.0 gp * * WS 3.0 a1 gp 200 4.0 gp br 50 300 600 (Base) C ol gp a1 -4 gp WS a1 a1 br -2 -2 i1br i1 -5 i1 -5 * * 100 gp gp WS a1 a1 br -2 -2 i1br i1 -5 gp Epidermal cell length (mm) 150 0 F 200 400 Number of epidermal cells 250 D 25 Epidermal cell length (mm) * 10 300 C 500 gp WS a1 a1 br -2 -2 i1br i1 -5 # E 350 t2 1.0 # * Maximal cell length (mm) * * 15 * * 5.0 -4 2.0 6.0 Hypocotyl length (mm) 3.0 gp 20 Average cell length (mm) D 25 * 4.0 br i1 -5 5.0 a1 -2 a1 -2 W S gp Number of epidermal cells C B A 6.0 de B Hypocotyl length (mm) A Average cell length (mm) 1018 Col gpa1-4 500 det2-1 400 gpa1-4 det2-1 300 200 100 (Base) 10 13 16 Cell number 19 21 (Top) Fig The number and length of hypocotyl epidermal cells in low-light-grown gpa1-2 bri1-5 double mutants (A) Phenotype of 4-day-old, low-light-grown seedlings Scale bar, mm Arrowheads point to the base and the top of hypocotyls (B) Hypocotyl length of 4-day-old, low-light-grown seedlings (C) Number of hypocotyl epidermal cells (D) The average lengths of hypocotyl epidermal cells in a single cell file (from base to top) (E) The maximal length of hypocotyl epidermal cells The three longest hypocotyl epidermal cells in each seedling were examined significant difference from WS, P50.05 #significant difference from bri1-5 mutant, P50.05 (F) The number and length of hypocotyl epidermal cells in a single cell file of gpa1-2 bri1-5 double mutants, compared with that in single mutants Shown in (B) are the averages of hypocotyl length from 20 seedlings for each genotype SE Shown in (C–F) are the averages of 10 seedlings for each genotype SE Fig The number and length of hypocotyl epidermal cells in low-light-grown gpa1-4 det2-1 double mutants (A) Phenotype of 4-day-old, low-light-grown seedlings Scale bar, mm Arrowheads point to the base and the top of hypocotyls (B) Hypocotyl length of 4-day-old, low-light-grown seedlings (C) Number of hypocotyl epidermal cells (D) The average lengths of hypocotyl epidermal cells in a single cell file (from base to top) (E) The maximal length of hypocotyl epidermal cells The three longest hypocotyl epidermal cells in each seedling were examined (F) The number and length of hypocotyl epidermal cells in a single cell file of gpa1-4 det2-1 double mutants, compared with that in single mutants significant difference from Col, P50.05 #significant difference from the det2-1 mutant, P50.05 Shown in (B) are the averages of hypocotyl length from 20 seedlings for each genotype SE Shown in (C–F) are the averages of 10 seedlings for each genotype SE Subsequently, we wanted to determine if such enhanced defects in root development caused by gpa1 mutations were also due to the enhanced defects in cell division Unlike hypocotyls in which the number of cells in the epidermis and cortex is pre-determined during embryogenesis and little cell division occurs in the epidermis or cortex, cell division occurs in various stages of root development Cell division in the root apical meristem and 1019 -2 C 80 60 * # * 40 20 Number of lateral roots 35 30 * 25 20 * 15 # * 10 -4 F E 30 60 50 40 * 30 # * 20 10 25 * 20 * 15 # * 10 -1 t2- t2 gp a1 -4 de -4 de Co a1 t2- -1 de t2 gp a1 -4 -4 a1 de l Co gp l 0 gp Length of primary root (mm) 70 Number of lateral roots a1 gp -1 t2 de gp Co l D a1 -4 de t2- gp gp W S a1 -2 br i1gp a1 -2 br i15 W S a1 -2 b ri1 gp -5 a1 -2 br i15 Length of primary root (mm) B gp a1 i1br W S A gp a1 -2 br i1 -5 Heterotrimeric G-proteins and brassinosteroid Fig Root phenotype of gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants (A) Phenotype of 7-day-old, light-grown gpa1-2 bri1-5 seedlings Scale bar, mm (B) Length of the primary root of gpa1-2 bri1-5 mutants (C) The number of lateral roots of gpa1-2 bri1-5 mutants (D) Phenotype of 7-day-old, light-grown gpa1-4 det2-1 seedlings (E) Length of the primary root of gpa1-4 det2-1 mutants (F) The number of lateral roots of gpa1-4 det2-1 mutants Shown in (B), (C), (E) and (F) are the averages of 10 seedlings for each genotype SE Data were collected from 10-day-old, light-grown seedlings significant difference from WS or Col, P50.05 #significant difference from the bri1-5 or det2-1 mutant, P50.05 cell elongation in the elongation zone determine the length of the primary root, whereas the formation of the lateral root requires the activation of or re-entry into the cell cycle in pericycle founder cells Therefore, a short root could be due to defects in cell division or cell elongation, or both The number of lateral roots is generally regarded as an indirect measurement of cell division in the pericycle founder cells, although initiation of cell division in the pericycle does not always lead to lateral root initiation (Vanneste et al 2005) However, because both bri1-5 and det2-1 mutants also have short roots, a direct comparison of the absolute number of lateral roots between single and double mutants may not be a reliable method for determining the relationship between gpa1 mutations and bri1-5 or det2-1 mutations in cell division during the process of lateral root formation Therefore, in this study, we specifically focused on the primary root to determine the cellular basis of the enhanced short root phenotypes of gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants We used a similar assay to that used previously to determine the cellular basis of altered length of primary roots in G-protein subunit and signaling component mutants (Chen et al 2003, Chen et al 2006) We measured the length of cortex cells in the root hair zone in which the 1020 Heterotrimeric G-proteins and brassinosteroid cortex cells have finished elongation The length of cortex cells in this region is generally used to reflect their ability for cell elongation (occurring in the cell elongation zone) The rate of cell production (number of cells h–1), which is generally used to reflect cell division activity in the root apical meristem, was calculated by using the rate of primary root growth (mm h–1) divided by the average length of mature cortex cells (mm) As reported previously (Chen et al 2006b), gpa1 mutants had the wild-type length of cortex cells and the wild-type rate of root cell production (Fig 7) While both bri1-5 and det2-1 mutants had shorter cortex cells (an indicator of cell elongation defect), compared with the wild type, they also had a reduced rate of 140 C 120 * * 100 80 60 40 20 Length of cortex cells (mm) 120 100 * * 80 60 40 20 1.0 * # * 0.6 0.4 0.2 Rate of root cell production (cell/h) D 0.8 -1 t2- de -4 a1 gp gp 1.2 0.9 * 0.6 # * 0.3 -1 de t21 -4 t2 a1 -4 de gp a1 br i1- gp a1 -2 -2 i1br S W a1 gp Co l 0 gp Rate of root cell productiion (cell/h) B t2 -4 gp a1 -2 de Co l 5 i1br i1- br gp a1 -2 W S 140 a1 Length of cortex cells (mm) A Fig Rate of cell production in the primary roots of gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants (A) Average length of mature root cortex cells in the gpa1-2 bri1-5 double mutants (B) Rate of cell production in the primary root of gpa1-2 bri1-5 double mutants (C) Average length of mature root cortex cells in the gpa1-4 det2-1 double mutants (D) Rate of cell production in the primary root of gpa1-4 det2-1 double mutants The rate of cell production was calculated as the rate of primary root elongation divided by the average length of mature cortical cells in the root hair zone Shown in (C) and (D) are the averages SE of 10 seedlings for each genotype significant difference from the wild type (WS or Col), P50.05 #significant difference from the bri1-5 or det2-1 mutant, P50.05 root cell production (an indicator of cell division defect) (Fig 7) Interestingly, the reduced rate of root cell production in the bri1-5 and det2-1 mutants was significantly enhanced by gpa1 mutations (Fig 7B, D) These results suggested that gpa1 mutations enhance the shortness of primary root of bri1-5 and det2-1 mutants by specifically enhancing their cell division defects Discussion Heterotrimeric G-protein complexes may serve as a nexus for the signal regulation of multiple developmental processes and hormonal responses in plants Loss-of-function mutations in G-protein subunits resulted in multiple morphological and conditional phenotypes (reviewed by Perfus-Barbeoch et al 2004, Chen 2008), and conferred defects in cell division throughout plant development (Ullah et al 2001, Chen et al 2003, Ullah et al 2003, Chen et al 2006) Loss-of-function alleles of Ga in Arabidopsis and in rice displayed altered sensitivities to BR (Ullah et al 2002, Wang et al 2006), suggesting that G-proteins are involved in the BR-mediated pathway Here we provide evidence that loss-of-function mutations in the sole Ga in Arabidopsis, GPA1, can specifically enhance the cell division defects of BR signaling and biosynthesis mutants We propose that GPA1- and BR-mediated pathways may act in concert to modulate cell proliferation in a cell/tissue-specific manner Role of tthe heterotrimeric G-protein a subunit in cell elongation and cell division GPA1 has been known as a critical modulator of cell division (Ullah et al 2001, Chen et al 2003, Ullah et al 2003, Chen et al 2006) A role for GPA1 in cell elongation has not been unequivocally established in Arabidopsis Cell division in the hypocotyl epidermal cells of gpa1 mutants is reduced, but cell elongation is unaffected (Figs 4, 5), supporting a prominent role for GPA1 in modulating cell division over cell elongation Because the length of epidermal cells along a hypocotyl appears to have a positional effect, with the longest cell located near the middle of a hypocotyl in both wild-type and gpa1 mutants under our assay conditions (Figs 4, 5), and because gpa1 mutants have fewer cells than the wild type, a direct cell to cell comparison may not reflect the overall difference in cell elongation between wild-type and gpa1 mutants Therefore, we chose to use the parameters of average cell length of all cells in a single cell file and the maximal cell length of the three longest cells in a single cell file to compare the difference in cell elongation among different genotypes We found that the average cell length and maximal cell length of hypocotyl epidermal cells in gpa1 mutants not differ significantly from those of wild-type seedlings (Figs 4, 5) Heterotrimeric G-proteins and brassinosteroid Interestingly, the rice Ga (RGA1) has been best known for its role in response to gibberellins (Fujisawa et al 1999, Ueguchi-Tanaka et al 2000) The dwarfism of the rice d1 mutant was caused by a loss-of-function mutation in the Ga gene (Fujisawa et al 1999) The d1 mutant also displayed reduced sensitivity to BR in the BR-mediated inhibition of root elongation, promotion of lamina inclination and promotion of coleoptile elongation (Wang et al 2006) More strikingly, d1 mutants are dwarf whereas gpa1 mutants have wild-type height It remains unclear why the loss of function of Ga in one species (e.g rice) results in dwarfism whereas it does not so in another species (e.g Arabidopsis) Role of BR in regulating cell elongation and cell division BR regulates both cell division and cell elongation, but is generally believed to have a prominent role in regulating cell elongation over cell division Both BR signaling mutants, such as bri1-5 (Li and Chory 1997, Noguchi et al 1999a) and bin2 (Li and Nam 2002), and BR biosynthesis mutants, such as det2-1 (Li et al 1996, Li et al 1997) and dwf4-102 (Azpiroz et al 1998, Choe et al 1998, Nakamoto et al 2006), displayed dwarfism Although a role for BR in cell division has been established in tissue culture and cell culture systems (Sala and Sala 1985, Nakajima et al 1996, Oh and Clouse 1998, Hu et al 2000, Miyazawa et al 2003), the genetic evidence had been lacking By using the Arabidopsis hypocotyl as a model system in which the number of epidermal cells is established during embryogenesis, we demonstrated that BR has a role in regulating both cell elongation and cell division We found that the length of hypocotyl epidermal cells was dramatically reduced in the BR receptor mutants, bri1-5 and bri1-4, and the BR biosynthesis mutants, det2-1 and dwf4-102 (Fig 2, Supplementary Fig S2), supporting the view that BR is a major regulator of cell elongation However, we found that the number of hypocotyl epidermal cells was also significantly reduced in these mutants (Fig 2, Supplementary Fig S2) These results provided direct evidence that BR regulates both cell elongation and cell division We extended our analysis to the roots Consistent with previous reports of BR mutants (Mouchel et al 2006), bri1-5 and det2-1 mutants had shorter primary roots than the wild type (Fig 6) By analyzing the length of root cortex cells and the rate of root cell production in bri1-5 and det2-1 mutants (Fig 7), we showed that BR also regulates both cell elongation and cell division in the roots Additive effect of GPA1 and BR in regulating cell division in hypocotyls We used the Arabidopsis hypocotyl as a model system to analyze the cellular basis of enhanced dwarfism phenotypes of gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants, 1021 originally observed in adult plants (Fig 1) Both gpa1 and BR mutants, bri1-5 and det2-1, share the short hypocotyl phenotype (Fig 2) However, the cellular basis of such shortness is different The short hypocotyl phenotype of gpa1 mutants was due to a reduction in cell division, whereas the short hypocotyl phenotype of bri1-5 and det2-1 mutants was due to a reduction in both cell elongation and cell division (Fig 2) Further, we found that gpa1 mutations could specifically enhance the cell division defects of bri1-5 and det2-1 mutants (Figs 4, 5) Because the cell division defects between gpa1-2 and bri1-5 and between gpa1-4 and det2-1 were additive, these results suggested that GPA1 and BR probably function in parallel pathways to regulate cell division in hypocotyls Similarly, GPA1 and BR may also function in independent pathways to regulate lateral root formation, because an additive effect was observed between gpa1-2 and bri1-5 and between gpa1-4 and det2-1 (Fig 6) Synergistic effect of GPA1 and BR in regulating cell division in roots As discussed above, GPA1 and BR probably function independently to regulate cell division in hypocotyls Interestingly, we found that GPA1 and BR may act in concert to regulate cell division in the primary roots (Figs 6, 7) gpa1 mutants have primary roots of normal length, whereas bri1-5 and det2-1 mutants have short primary roots (Fig 6) We found that the short root phenotype of bri1-5 and det2-1 mutants was due to a defect in both cell elongation and cell division (Fig 7) Interestingly, we found that although gpa1 mutants not display defects in cell elongation or cell division in the primary roots, gpa1 mutations can specifically enhance the cell division defects in the primary roots of bri1-5 and det2-1 mutants (Fig 7) Such s synergistic effect suggested that GPA1 may interact genetically with the BR-mediated pathways to regulate cell division in the primary roots Similarly, GPA1- and BR-mediated pathways may work together to regulate plant height because gpa1 mutants have wild-type height but can significantly enhance the dwarfism of bri1-5 and det2-1 mutants (Fig 1) These findings raise the possibility that the G-proteinmediated pathway may cross-talk with the receptor kinasemediated pathway to regulate a specific cellular process (e.g cell division) in plants How can GPA1- and BR-mediated pathways function independently in one tissue (e.g hypocotyl) whereas they work together in other tissues (e.g primary root) to regulate cell division? One crucial concept of heterotrimeric G-protein action in plants is that G-proteins play regulatory roles in diverse developmental processes and function in a cell type- or developmental stage-specific manner (Perfus-Barbeoch et al 2004) For example, gpa1 mutants are hypersensitive to ABA during seed germination and early seedling development (Ullah et al 2002, 1022 Heterotrimeric G-proteins and brassinosteroid Pandey et al 2006) whereas they are insensitive to ABA in guard cells by abolishing the inhibition of the inward Kỵ channels by ABA (Wang et al 2001) Another example is that the sole Arabidopsis heterotrimeric G-protein b subunit, AGB1, has been shown to be a positive regulator of axial cell division in the hypocotyls, whereas it functions as a negative regulator of cell division in the roots (Ullah et al 2003, Chen et al 2006) It is likely that G-proteins may utilize a signaling cascade to regulate cell division in the primary roots that is distinct from that in the hypocotyls The exact molecular mechanisms of GPA1 and BR in regulating cell division are presently unknown gpa1 mutants displayed altered sensitivities to multiple hormones, such as auxin (Ullah et al 2003), ABA (Wang et al 2001, Ullah et al 2002, Pandey et al 2006) and gibberellins (Ullah et al 2002, Chen et al 2004), in addition to BR (Ullah et al 2002) On the other hand, BR also crosstalks with other hormones, particularly auxin (Nemhauser et al 2004, Hardtke 2007) For example, BR can activate the expression of some auxin-induced genes, as revealed by microarray analyses (Goda et al 2004, Nemhauser et al 2004), and act synergistically with auxin to regulate hypocotyl growth (Nakamura et al 2003, Nemhauser et al 2004) and lateral root formation (Bao et al 2004) in Arabidopsis Recently, BREVIS RADIX (BRX) has been shown to act at the nexus of a feedback loop that maintains threshold BR levels to permit optimal auxin action in roots (Mouchel et al 2006) These findings raise the possibility that GPA1and BR-mediated pathways may interact through interaction with other plant hormones However, because a canonical GPCR together with its ligand has yet to be identified in plants and the G-protein-mediated signal transduction pathway in plants is far from complete, the elucidation of the interaction between G-proteins and the BR signaling pathway at the mechanistic level is a challenging yet exciting topic for future research Materials and Methods Plant materials gpa1-2 (Ullah et al 2001), bri1-5 (Li and Chory 1997, Noguchi et al 1999a) and bri1-4 (Li and Chory 1997, Noguchi et al 1999a) mutants are in the WS ecotypic background gpa1-4 (Jones et al 2003), det2-1 (Li and Chory 1996, Li et al 1997) and dwf4-102 (Azpiroz et al 1998, Choe et al 1998, Nakamoto et al 2006) are in the Col ecotypic background gpa1-2 bri1-5 double mutants were generated by crossing gpa1-2 (pollen donor) into bri1-5 and identified from the F2 progeny by PCR genotyping and phenotyping Similarly, gpa1-4 det2-1 double mutants were generated by crossing gpa1-4 into det2-1 and identified from the F2 progeny Plant growth conditions and phenotypic analyses For Petri dish-based phenotypic analyses, wild-type and mutant seeds were sterilized, sown on MS/G Petri dishes containing 1/2 strength Murashige and Skoog (MS) basal medium with vitamins (plantmedia, Dublin, OH, USA), 1% sucrose and 0.5% phytoagar (plantmedia), adjusted to pH 5.7 with N KOH, and treated at 48C in the dark for d, then moved to a growth chamber at 238C For the phenotypic analysis of 3-day-old, dark-grown seedlings, the Petri dishes were wrapped in double layers of aluminum foil and placed in the dark For light-grown seedlings, Petri dishes were placed under low-light conditions (30 mmol m–2 s–1) with a 14/10 h photoperiod for easy examination of hypocotyl epidermal and cortex cells The hypocotyl lengths were measured from at least 20 seedlings for each genotype For soil-based phenotypic analysis, wild-type and mutant plants were grown under identical conditions with a 14/10 h photoperiod at 140 mmol m–2 s–1 Plant heights were measured from at least 10 plants for each genotype Measurement of hypocotyl cells For the examination of hypocotyl epidermal cells and outer cortex cells, 3-day-old, dark-grown, or 4-day-old, low-lightgrown seedlings of the wild type and mutants were fixed and cleared in chloral hydrate solution (chloral hydrate : glycerol : water ¼ : : 3) A single file of epidermal or outer cortex cells from the base (root–hypocotyl junction) to the top of the hypocotyl (lateral to cotyledons) of each seedling was examined under a Leica DM-6000B upright microscope with phase and DIC equipped with a Leica FW4000 digital image acquisition and processing system [Leica Microsystems (Canada) Inc., Richmond Hill, Ontario, Canada] Root assays Seedlings grown on MS/G plates were placed under a 14/10 h photoperiod with approximately 120 mmol m–2 s–1 at 238C with a vertical orientation for monitoring root growth The length of primary roots and the number of lateral roots were determined on 10-day-old seedlings, and at least 10 seedlings were used for each genotype The procedure for the measurement of the rate of root cell production has been described previously (Chen et al 2003, Chen et al 2006) The root growth was monitored daily by marking the positions of the root tips Rates of primary root growth were calculated over d periods from day to day Seedlings were sampled at day 7, fixed, and cleared in chloral hydrate solution The lengths of at least 20 cortex cells in the root hair zone of each root were measured under a Leica DM-6000B upright microscope with phase and DIC equipped with a Leica FW4000 digital image acquisition and processing system Cell production was calculated as the rate of root growth divided by the average length of mature cortex cells Supplementary material Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oxford journals.org Funding Natural Sciences and Engineering Research Council of Canada and Canada Foundation for Innovation (to J.-G.C.) 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