The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light

Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4). Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids. Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness. While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light. Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling. Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light. Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A. Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1. Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1. Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.     

ATH1 and KNAT2 proteins act together in regulation of plant inflorescence architecture

The inflorescence of flowering plants is a highly organized structure, not only contributing to plant reproductive processes, but also constituting an important part of the entire plant morphology. Previous studies have revealed that the class-I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP or KNAT1), KNAT2, and KNAT6 play essential roles in inflorescence architecture. Pedicel morphology is known to contribute greatly to inflorescence architecture, and BP negatively regulates KNAT2 and KNAT6 to ensure that pedicels have a normal upward-pointing orientation. These findings indicate that a genetic network exists in controlling pedicel orientation, but how this network functions in the developmental process remains elusive. Here it is reported that the ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1) gene, which belongs to the BELL1-like homeodomain gene family, is a new member participating in regulating pedicel orientation in the class-I KNOX network. In a genetic screening for suppressors of isoginchaku-2D, a gain-of-function ASYMMETRIC LEAVES2 mutant that displays downward-pointing pedicels, a suppressor mutant was obtained. Characterization of this mutant revealed that the mutation corresponds to ATH1. Genetic analysis indicated that ATH1 acts mainly in the KNAT2 pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that ATH1 physically interacts with KNAT2. The data indicate that the ATH1-KNAT2 complex acts redundantly with KNAT6, both of which are negatively regulated by BP during pedicel development.     

Arl8 and SKIP act together to link lysosomes to kinesin-1

Lysosomes move bidirectionally on microtubules, and this motility can be stimulated by overexpression of the small GTPase Arl8. By using affinity chromatography, we find that Arl8-GTP binds to the soluble protein SKIP (SifA and kinesin-interacting protein, aka PLEKHM2). SKIP was originally identified as a target of the Salmonella effector protein SifA and found to bind the light chain of kinesin-1 to activate the motor on the bacteria's replicative vacuole. We show that in uninfected cells both Arl8 and SKIP are required for lysosomes to distribute away from the microtubule-organizing center. We identify two kinesin light chain binding motifs in SKIP that are required for lysosomes to accumulate kinesin-1 and redistribute to the cell periphery. Thus, Arl8 binding to SKIP provides a link from lysosomal membranes to plus-end-directed motility. A splice variant of SKIP that lacks a light chain binding motif does not stimulate movement, suggesting fine-tuning by alternative splicing.     

BAS1 and SOB7 act redundantly to modulate Arabidopsis photomorphogenesis via unique brassinosteroid inactivation mechanisms

Active brassinosteroids (BRs), such as brassinolide (BL) and castasterone (CS), are growth-promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase (CYP734A1, formerly CYP72B1), encoded by the BAS1 gene, inactivates BRs and modulates photomorphogenesis. BAS1 was identified as the overexpressed gene responsible for a dominant, BR-deficient mutant, bas1-D. This mutant was isolated in an activation-tagged screen designed to identify redundant genes that might not be identified in classic loss-of-function screens. Here we report the isolation of a second activation-tagged mutant with a BR-deficient phenotype. The mutant phenotype is caused by the overexpression of SOB7 (CYP72C1), a homolog of BAS1. We generated single and double null-mutants of BAS1 and SOB7 to test the hypothesis that these two genes act redundantly to modulate photomorphogenesis. BAS1 and SOB7 act redundantly with respect to light promotion of cotyledon expansion, repression of hypocotyl elongation and flowering time in addition to other phenotypes not regulated by light. We also provide biochemical evidence to suggest that BAS1 and SOB7 act redundantly to reduce the level of active BRs, but have unique mechanisms. Overexpression of SOB7 results in a dramatic reduction in endogenous CS levels, and although single null-mutants of BAS1 and SOB7 have the same level of CS as the wild type, the double null-mutant has twice the amount. Application of BL to overexpression lines of BAS1 or SOB7 results in enhanced metabolism of BL, though only BAS1 overexpression lines confer enhanced conversion to 26-OHBL, suggesting that SOB7 and BAS1 convert BL and CS into unique products.     

Two subunits of the Drosophila mediator complex act together to control cell affinity

The organizing centers for Drosophila imaginal disc development are created at straight boundaries between compartments; these are maintained by differences in cell affinity controlled by selector genes and intercellular signals. skuld and kohtalo encode homologs of TRAP240 and TRAP230, the two largest subunits of the Drosophila mediator complex; mutations in either gene cause identical phenotypes. We show here that both genes are required to establish normal cell affinity differences at the anterior-posterior and dorsal-ventral compartment boundaries of the wing disc. Mutant cells cross from the anterior to the posterior compartment, and can distort the dorsal-ventral boundary in either the dorsal or ventral direction. The Skuld and Kohtalo proteins physically interact in vivo and have synergistic effects when overexpressed, consistent with a skuld kohtalo double-mutant phenotype that is indistinguishable from either single mutant. We suggest that these two subunits do not participate in all of the activities of the mediator complex, but form a submodule that is required to regulate specific target genes, including those that control cell affinity.     

SRC-1 and Wnt signaling act together to specify endoderm and to control cleavage orientation in early C. elegans embryos

In early C. elegans embryos, signaling between a posterior blastomere, P2, and a ventral blastomere, EMS, specifies endoderm and orients the division axis of the EMS cell. Although Wnt signaling contributes to this polarizing interaction, no mutants identified to date abolish P2/EMS signaling. Here, we show that two tyrosine kinase-related genes, src-1 and mes-1, are required for the accumulation of phosphotyrosine between P2 and EMS. Moreover, src-1 and mes-1 mutants strongly enhance endoderm and EMS spindle rotation defects associated with Wnt pathway mutants. SRC-1 and MES-1 signal bidirectionally to control cell fate and division orientation in both EMS and P2. Our findings suggest that Wnt and Src signaling function in parallel to control developmental outcomes within a single responding cell.     

Wnt and EGF pathways act together to induce C. elegans male hook development

Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1°, 2°, 3°), and 2° HCG fate specification is mediated by LIN-12/Notch. We show that 2° HCG specification depends on the presence of a cell with the 1° fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 acts to specify the 1° and 2° HCG fate. A requirement for EGF signaling during 1° fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that WNT plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or WNT induce a 1° lineage, and LIN-12/NOTCH induces a 2° lineage. Wnt signaling is also required for execution of the 1° and 2° HCG lineages. lin-17 and bar-1/β-catenin are preferentially expressed in the presumptive 1° cell P11.p. The dynamic subcellular localization of BAR-1-GFP in P11.p is concordant with the timing of HCG fate determination.     

FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.     

Mineralocorticoids and cerebral angiotensin may act together to produce sodium appetite

The sodium appetite that follows sodium deficiency may be aroused by a synergy of the hormones of sodium deficiency (angiotensin and mineralocorticoid) rather than by the deficiency itself. Recent evidence supporting this idea is discussed with emphasis on the possibility that angiotensin of cerebral origin may be more effective in this synergy than that of renal origin.     

KIF14 and citron kinase act together to promote efficient cytokinesis

Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735-1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877-885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175-1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711-5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832-1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237-3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115-127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.     

Mutations in the N‐terminal kinase‐like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark‐grown plants. While both COP1 and the four SPA proteins contain coiled‐coil and WD‐repeat domains, SPA proteins differ from COP1 in carrying an N‐terminal kinase‐like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N‐terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N‐terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N‐terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase‐like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase‐like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase‐like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N‐terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N‐terminal domain. Together, these results uncover an important, but complex role of the SPA1 N‐terminus in the suppression of photomorphogenesis.     

DDB2, DDB1A and DET1 exhibit complex interactions during Arabidopsis development

Damaged DNA-binding proteins 1 and 2 (DDB1 and DDB2) are subunits of the damaged DNA-binding protein complex (DDB). DDB1 is also found in the same complex as DE-ETIOLATED 1 (DET1), a negative regulator of light-mediated responses in plants. Arabidopsis has two DDB1 homologs, DDB1A and DDB1B. ddb1a single mutants have no visible phenotype while ddb1b mutants are lethal. We have identified a partial loss-of-function allele of DDB2. To understand the genetic interaction among DDB2, DDB1A, and DET1 during Arabidopsis light signaling, we generated single, double, and triple mutants. det1 ddb2 partially enhances the short hypocotyl and suppresses the high anthocyanin content of dark-grown det1 and suppresses the low chlorophyll content, early flowering time (days), and small rosette diameter of light-grown det1. No significant differences were observed between det1 ddb1a and det1 ddb1a ddb2 in rosette diameter, dark hypocotyl length, and anthocyanin content, suggesting that these are DDB1A-dependent phenotypes. In contrast, det1 ddb1a ddb2 showed higher chlorophyll content and later flowering time than det1 ddb1a, indicating that these are DDB1A-independent phenotypes. We propose that the DDB1A-dependent phenotypes indicate a competition between DDB2- and DET1-containing complexes for available DDB1A, while, for DDB1A-independent phenotypes, DDB1B is able to fulfill this role.     

Mammalian GGAs act together to sort mannose 6-phosphate receptors

The GGAs (Golgi-localized, gamma ear-containing, ADP ribosylation factor-binding proteins) are multidomain proteins implicated in protein trafficking between the Golgi and endosomes. We examined whether the three mammalian GGAs act independently or together to mediate their functions. Using cryo-immunogold electron microscopy, the three GGAs were shown to colocalize within coated buds and vesicles at the trans-Golgi network (TGN) of HeLa cells. In vitro binding experiments revealed multidomain interactions between the GGAs, and chemical cross-linking experiments demonstrated that GGAs 1 and 2 form a complex on Golgi membranes. RNA interference of each GGA resulted in decreased levels of the other GGAs and their redistribution from the TGN to cytosol. This was associated with impaired incorporation of the cation-independent mannose 6-phosphate receptor into clathrin-coated vesicles at the TGN, partial redistribution of the receptor to endosomes, and missorting of cathepsin D. The morphology of the TGN was also altered. These findings indicate that the three mammalian GGAs cooperate to sort cargo and are required for maintenance of TGN structure.     

Flowering time genes Heading date 1 and Early heading date 1 together control panicle development in rice

Although flowering time is often associated with plant size, little is known about how flowering time genes affect plant architecture. We grew four rice lines having different flowering time genotypes (hd1 ehd1, hd1 Ehd1, Hd1 ehd1 and Hd1 Ehd1) under distinct photoperiod conditions. By using genotype-treatment combinations that resulted in similar flowering times, we were able to compare the effects of flowering time genes on traits related to plant architecture. The results revealed that the combination of Heading-date 1 (Hd1) and Early heading date 1 (Ehd1) can reduce the number of primary branches in a panicle, resulting in smaller spikelet numbers per panicle; this occurs independently of the control of flowering time. In addition, expression of the Hd3a and Rice Flowering-locus T 1 (RFT1) florigen genes was up-regulated in leaves of the Hd1 Ehd1 line at the time of the floral transition. We further revealed that Hd1 and/or Ehd1 caused up-regulation of Terminal Flower 1-like genes and precocious expression of panicle formation-related genes at shoot apical meristems during panicle development. Therefore, two key flowering time genes, Hd1 and Ehd1, can control panicle development in rice; this may affect crop yields in the field through florigen expression in leaf.