Nature and Nurture: Genotype-Dependent Differential Responses of Root Architecture to Agar and Soil Environments.

Root development is crucial for plant growth and therefore a key factor in plant performance and food production. Arabidopsis thaliana is the most commonly used system to study root system architecture (RSA). Growing plants on agar-based media has always been routine practice, but this approach poorly reflects the natural situation, which fact in recent years has led to a dramatic shift toward studying RSA in soil. Here, we directly compare RSA responses to agar-based medium (plates) and potting soil (rhizotrons) for a set of redundant loss-of-function plethora (plt) CRISPR mutants with variable degrees of secondary root defects. We demonstrate that plt3plt7 and plt3plt5plt7 plants, which produce only a handful of emerged secondary roots, can be distinguished from other genotypes based on both RSA shape and individual traits on plates and rhizotrons. However, in rhizotrons the secondary root density and the total contribution of the side root system to the RSA is increased in these two mutants, effectively rendering their phenotypes less distinct compared to WT. On the other hand, plt3, plt3plt5, and plt5plt7 mutants showed an opposite effect by having reduced secondary root density in rhizotrons. This leads us to believe that plate versus rhizotron responses are genotype dependent, and these differential responses were also observed in unrelated mutants short-root and scarecrow. Our study demonstrates that the type of growth system affects the RSA differently across genotypes, hence the optimal choice of growth conditions to analyze RSA phenotype is not predetermined.

The Floral C-Lineage Genes Trigger Nectary Development in Petunia and Arabidopsis.

To attract insects, flowers produce nectar, an energy-rich substance secreted by specialized organs called nectaries. For Arabidopsis thaliana, a rosid species with stamen-associated nectaries, the floral B-, C-, and E-functions were proposed to redundantly regulate nectary development. Here, we investigated the molecular basis of carpel-associated nectary development in the asterid species petunia (Petunia hybrida). We show that its euAGAMOUS (euAG) and PLENA (PLE) C-lineage MADS box proteins are essential for nectary development, while their overexpression is sufficient to induce ectopic nectaries on sepals. Furthermore, we demonstrate that Arabidopsis nectary development also fully depends on euAG/PLE C-lineage genes. In turn, we show that petunia nectary development depends on two homologs of CRABS CLAW (CRC), a gene previously shown to be required for Arabidopsis nectary development, and demonstrate that CRC expression in both species depends on the members of both euAG/PLE C-sublineages. Therefore, petunia and Arabidopsis employ a similar molecular mechanism underlying nectary development, despite otherwise major differences in the evolutionary trajectory of their C-lineage genes, their distant phylogeny, and different nectary positioning. However, unlike in Arabidopsis, petunia nectary development is position independent within the flower. Finally, we show that the TARGET OF EAT-type BLIND ENHANCER and APETALA2-type REPRESSOR OF B-FUNCTION genes act as major regulators of nectary size.

The tomato MAX1 homolog, SlMAX1, is involved in the biosynthesis of tomato strigolactones from carlactone.

Strigolactones (SLs) are rhizosphere signalling molecules exuded by plants that induce seed germination of root parasitic weeds and hyphal branching of arbuscular mycorrhiza. They are also phytohormones regulating plant architecture. MORE AXILLARY GROWTH 1 (MAX1) and its homologs encode cytochrome P450 (CYP) enzymes that catalyse the conversion of the strigolactone precursor carlactone to canonical strigolactones in rice (Oryza sativa), and to an SL-like compound in Arabidopsis. Here, we characterized the tomato (Solanum lycopersicum) MAX1 homolog, SlMAX1. The targeting induced local lesions in genomes method was used to obtain Slmax1 mutants that exhibit strongly reduced production of orobanchol, solanacol and didehydro-orobanchol (DDH) isomers. This results in a severe strigolactone mutant phenotype in vegetative and reproductive development. Transient expression of SlMAX1 - together with SlD27, SlCCD7 and SlCCD8 - in Nicotiana benthamiana showed that SlMAX1 catalyses the formation of carlactonoic acid from carlactone. Plant feeding assays showed that carlactone, but not 4-deoxy-orobanchol, is the precursor of orobanchol, which in turn is the precursor of solanacol and two of the three DDH isomers. Inhibitor studies suggest that a 2-oxoglutarate-dependent dioxygenase is involved in orobanchol biosynthesis from carlactone and that the formation of solanacol and DDH isomers from orobanchol is catalysed by CYPs.

The interaction of strigolactones with abscisic acid during the drought response in rice.

Both strigolactones (SLs) and abscisic acid (ABA) biosynthetically originate from carotenoids. Considering their common origin, the interaction of these two hormones at the biosynthetic and/or regulatory level may be anticipated. Here we show that, in rice, drought simultaneously induces SL production in the root, and ABA production and the expression of SL biosynthetic genes in the shoot. Under control conditions, the ABA concentration was higher in shoots of the SL biosynthetic rice mutants dwarf10 (d10) and d17 than in wild-type plants, while a similar trend was observed for the SL perception mutant d3. These differences were enhanced under drought. However, drought did not result in an increase in leaf ABA content in the rice mutant line d27, carrying a mutation in the gene encoding the first committed enzyme in SL biosynthesis, to the same extent as in the other SL mutants and the wild type. Accordingly, d10, d17, and d3 lines were more drought tolerant than wild-type plants, whereas d27 displayed decreased tolerance. Finally, overexpression of OsD27 in rice resulted in increased levels of ABA when compared with wild-type plants. We conclude that the SL and ABA pathways are connected with each other through D27, which plays a crucial role in determining ABA and SL content in rice.

Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis.

The flowers of most dicotyledons have petals that, together with the sepals, initially protect the reproductive organs. Later during development petals are required to open the flower and to attract pollinators. This diverse set of functions demands tight temporal and spatial regulation of petal development. We studied the functioning of the Arabidopsis thaliana TCP5-like transcription factors (TFs) in petals. Overexpression of TCP5 in petal epidermal cells results in smaller petals, whereas tcp5 tcp13 tcp17 triple knockout lines have wider petals with an increased surface area. Comprehensive expression studies revealed effects of TCP5-like TFs on the expression of genes related to the cell cycle, growth regulation and organ growth. Additionally, the ethylene biosynthesis genes 1-amino-cyclopropane-1-carboxylate (ACC) synthase 2 (ACS2) and ACC oxidase 2 (ACO2) and several ETHYLENE RESPONSE FACTORS (ERFs) are found to be differentially expressed in TCP5 mutant and overexpression lines. Chromatin immunoprecipitation-quantitative PCR showed direct binding of TCP5 to the ACS2 locus in vivo. Ethylene is known to influence cell elongation, and the petal phenotype of the tcp5 tcp13 tcp17 mutant could be complemented by treatment of the plants with an ethylene pathway inhibitor. Taken together, this reveals a novel role for TCP5-like TFs in the regulation of ethylene-mediated petal development and growth.

Analysis of functional redundancies within the Arabidopsis TCP transcription factor family.

Analyses of the functions of TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1 (TCP) transcription factors have been hampered by functional redundancy between its individual members. In general, putative functionally redundant genes are predicted based on sequence similarity and confirmed by genetic analysis. In the TCP family, however, identification is impeded by relatively low overall sequence similarity. In a search for functionally redundant TCP pairs that control Arabidopsis leaf development, this work performed an integrative bioinformatics analysis, combining protein sequence similarities, gene expression data, and results of pair-wise protein-protein interaction studies for the 24 members of the Arabidopsis TCP transcription factor family. For this, the work completed any lacking gene expression and protein-protein interaction data experimentally and then performed a comprehensive prediction of potential functional redundant TCP pairs. Subsequently, redundant functions could be confirmed for selected predicted TCP pairs by genetic and molecular analyses. It is demonstrated that the previously uncharacterized class I TCP19 gene plays a role in the control of leaf senescence in a redundant fashion with TCP20. Altogether, this work shows the power of combining classical genetic and molecular approaches with bioinformatics predictions to unravel functional redundancies in the TCP transcription factor family.

Arabidopsis class I and class II TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) transcription factors control developmental processes in plants. The 24 TCP transcription factors encoded in the Arabidopsis (Arabidopsis thaliana) genome are divided into two classes, class I and class II TCPs, which are proposed to act antagonistically. We performed a detailed phenotypic analysis of the class I tcp20 mutant, showing an increase in leaf pavement cell sizes in 10-d-old seedlings. Subsequently, a glucocorticoid receptor induction assay was performed, aiming to identify potential target genes of the TCP20 protein during leaf development. The LIPOXYGENASE2 (LOX2) and class I TCP9 genes were identified as TCP20 targets, and binding of TCP20 to their regulatory sequences could be confirmed by chromatin immunoprecipitation analyses. LOX2 encodes for a jasmonate biosynthesis gene, which is also targeted by class II TCP proteins that are under the control of the microRNA JAGGED AND WAVY (JAW), although in an antagonistic manner. Mutation of TCP9, the second identified TCP20 target, resulted in increased pavement cell sizes during early leaf developmental stages. Analysis of senescence in the single tcp9 and tcp20 mutants and the tcp9tcp20 double mutants showed an earlier onset of this process in comparison with wild-type control plants in the double mutant only. Both the cell size and senescence phenotypes are opposite to the known class II TCP mutant phenotype in JAW plants. Altogether, these results point to an antagonistic function of class I and class II TCP proteins in the control of leaf development via the jasmonate signaling pathway.

SIN-inhibitory phosphatase complex promotes Cdc11p dephosphorylation and propagates SIN asymmetry in fission yeast.

BACKGROUND: Cytokinesis in many eukaryotes involves the function of an actomyosin-based contractile ring. In fission yeast, actomyosin ring maturation and stability require a conserved signaling pathway termed the SIN (septation initiation network). The SIN consists of a GTPase (Spg1p) and three protein kinases, all of which localize to the mitotic spindle pole bodies (SPBs). Two of the SIN kinases, Cdc7p and Sid1p, localize asymmetrically to the newly duplicated SPB in late anaphase. How this asymmetry is achieved is not understood, although it is known that their symmetric localization impairs cytokinesis. RESULTS: Here we characterize a new Forkhead-domain-associated protein, Csc1p, and identify SIN-inhibitory PP2A complex (SIP), which is crucial for the establishment of SIN asymmetry. Csc1p localizes to both SPBs early in mitosis, is lost from the SPB that accumulates Cdc7p, and instead accumulates at the SPB lacking Cdc7p. Csc1p is required for the dephosphorylation of the SIN scaffolding protein Cdc11p and is thereby required for the recruitment of Byr4p, a component of the GTPase-activating subunit for Spg1p, to the SPB. CONCLUSIONS: Because Cdc7p does not bind to GDP-Spg1p, we propose that the SIP-mediated Cdc11p dephosphorylation and the resulting recruitment of Byr4p are among the earliest steps in the establishment of SIN asymmetry.

A role for the septation initiation network in septum assembly revealed by genetic analysis of sid2-250 suppressors.

In the fission yeast Schizosaccharomyces pombe the septation initiation network (SIN) is required for stabilization of the actomyosin ring in late mitosis as well as for ring constriction and septum deposition. In a genetic screen for suppressors of the SIN mutant sid2-250, we isolated a mutation, ace2-35, in the transcription factor Ace2p. Both ace2Delta and ace2-35 show defects in cell separation, and both can rescue the growth defects of some SIN mutants at low restrictive temperatures, where the SIN single mutants lyse at the time of cytokinesis. By detailed analysis of the formation and constriction of the actomyosin ring and septum in the sid2-250 mutant at low restrictive temperatures, we show that the lysis phenotype of the sid2-250 mutant is likely due to a weak cell wall and septum combined with enzymatic activity of septum-degrading enzymes. Consistent with the recent findings that Ace2p controls transcription of genes involved in cell separation, we show that disruption of some of these genes can also rescue sid2-250 mutants. Consistent with SIN mutants having defects in septum formation, many SIN mutants can be rescued at the low restrictive temperature by the osmotic stabilizer sorbitol. The small GTPase Rho1 is known to promote cell wall formation, and we find that Rho1p expressed from a multi-copy plasmid can also rescue sid2-250 at the low restrictive temperature. Together these results suggest that the SIN has a role in promoting proper cell wall formation at the division septa.

Systematic deletion analysis of fission yeast protein kinases.

Eukaryotic protein kinases are key molecules mediating signal transduction that play a pivotal role in the regulation of various biological processes, including cell cycle progression, cellular morphogenesis, development, and cellular response to environmental changes. A total of 106 eukaryotic protein kinase catalytic-domain-containing proteins have been found in the entire fission yeast genome, 44% (or 64%) of which possess orthologues (or nearest homologues) in humans, based on sequence similarity within catalytic domains. Systematic deletion analysis of all putative protein kinase-encoding genes have revealed that 17 out of 106 were essential for viability, including three previously uncharacterized putative protein kinases. Although the remaining 89 protein kinase mutants were able to form colonies under optimal growth conditions, 46% of the mutants exhibited hypersensitivity to at least 1 of the 17 different stress factors tested. Phenotypic assessment of these mutants allowed us to arrange kinases into functional groups. Based on the results of this assay, we propose also the existence of four major signaling pathways that are involved in the response to 17 stresses tested. Microarray analysis demonstrated a significant correlation between the expression signature and growth phenotype of kinase mutants tested. Our complete microarray data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/kinome.

Roles of Pdk1p, a fission yeast protein related to phosphoinositide-dependent protein kinase, in the regulation of mitosis and cytokinesis.

Proteins related to the phosphoinositide-dependent protein kinase family have been identified in the majority of eukaryotes. Although much is known about upstream mechanisms that regulate the PDK1-family of kinases in metazoans, how these kinases regulate cell growth and division remains unclear. Here, we characterize a fission yeast protein related to members of this family, which we have termed Pdk1p. Pdk1p localizes to the spindle pole body and the actomyosin ring in early mitotic cells. Cells deleted for pdk1 display multiple defects in mitosis and cytokinesis, all of which are exacerbated when the function of fission yeast polo kinase, Plo1p, is partially compromised. We conclude that Pdk1p functions in concert with Plo1p to regulate multiple processes such as the establishment of a bipolar mitotic spindle, transition to anaphase, placement of the actomyosin ring and proper execution of cytokinesis. We also present evidence that the effects of Pdk1p on cytokinesis are likely mediated via the fission yeast anillin-related protein, Mid1p, and the septation initiation network.

The nuclear kinase Lsk1p positively regulates the septation initiation network and promotes the successful completion of cytokinesis in response to perturbation of the actomyosin ring in Schizosaccharomyces pombe.

Cytokinesis in fission yeast requires the function of an actomyosin-based contractile ring whose constriction is dependent on a signaling module termed the septation initiation network (SIN). In response to minor perturbation of the ring, the duration of SIN signaling is extended concurrently with a delay in nuclear cycle progression. These mechanisms require the conserved phosphatase Clp1p/Flp1p and facilitate the successful completion of cytokinesis, thereby increasing cellular viability. To isolate novel components of this cytokinesis monitoring system, we screened a genome-wide bank of protein kinase deletion mutants and identified Lsk1p, a nuclear-localized protein kinase. Similar to clp1Delta mutants, and in contrast to wild type, lsk1Delta cells are unable to maintain the integrity of the actomyosin ring upon treatment with low doses (0.3 microM) of latrunculin A. However, unlike clp1Delta mutants, lsk1Delta cells are competent to delay nuclear cycle progression after cytokinetic failure. In addition, lsk1Delta mutants suppress the lethal, multiseptate phenotype conferred by hyperactivation of the SIN, demonstrating that Lsk1p is a positive regulator of this module. In this report, we demonstrate that Lsk1p acts in parallel to Clp1p to promote actomyosin ring stability upon checkpoint activation. Our studies also establish that actomyosin ring maintenance and nuclear cycle delay in response to cytokinetic perturbation can be genetically resolved into independent pathways.

A potential tension-sensing mechanism that ensures timely anaphase onset upon metaphase spindle orientation.

The spindle orientation checkpoint (SOC) in fission yeast has been proposed to delay metaphase-to-anaphase transition when the spindle poles are misaligned with respect to the long axis of the cell. This checkpoint is activated in the absence of either an actomyosin division ring or astral microtubules. Although the SOC could be overridden in the absence of the transcription factor Atf1p, its mechanistic nature remained unclear. Here, we show that the SOC-triggered metaphase delay depends on a subset of the spindle assembly checkpoint (SAC) components Mph1p and Bub1p. Based on this finding and a detailed imaging of the spindle orientation process, we hypothesized that the spindle pole might contain proteins capable of sensing the achievement of spindle alignment. We identified the kendrin-like spindle pole body resident Pcp1p as a candidate molecule. A targeted mutation in its central domain specifically triggered the SOC in spite of the presence of oriented spindles, causing a metaphase delay that could be relieved in the absence of Mph1p, Bub1p, and Atf1p. Thus, Pcp1p might provide a link between the mechanical process of spindle alignment and the signal transduction that initiates anaphase.

Functional diversity of potato SNF1-related kinases tested in Saccharomyces cerevisiae.

Sucrose nonfermenting 1 catalytic subunit (SNF1)-type protein kinases are members of a metabolite-sensing protein kinase family distributed ubiquitously from yeast to plants and animals. In yeast cells, SNF1 acts in complex with the activator subunit SNF4 and a member of the SIP1/SIP2/GAL83 family responsible for substrate definition. The potato (Solanum tuberosum) genome possesses at least two SnRK1s, designated PKIN1 and StubSNF1. In this study, potato kinase 1 (PKIN1) and StubSNF1 were analysed in the yeast two-hybrid system and characterised by suppression of yeast mutations. It was shown that StubSNF1 interacted with the GAL83 ortholog of potato, StubGAL83, and complemented the Delta snf1 mutation. Moreover, it suppressed Delta snf4 and Delta sip1,Delta sip2,Delta gal83 deficiencies. In contrast, PKIN1 was unable to interact with StubGAL83 and did not rescue the yeast mutants. These data suggest different functions for PKIN1 and StubSNF1 in potato.

Antisense repression of StubGAL83 affects root and tuber development in potato.

StubGAL83 is a potato gene that encodes the beta-subunit of a protein kinase complex similar to the yeast SNF1, and the mammalian AMPK complexes that are modulated by changes in the cellular AMP/ATP ratio and are important regulators of metabolic and stress responses. Here we show that the expression of StubGAL83 in potato foliage is much higher in the dark than in the light and can be repressed by metabolisable sugars in the dark. The amounts of StubGAL83 mRNA are higher in sink than in source leaves. To unravel the role of StubGAL83, transgenic potato plants expressing a part of the StubGAL83 cDNA in antisense orientation under the control of the constitutive CaMV35S promoter were generated. Northern analysis revealed a reduction up to 90-95% in StubGAL83 mRNA accumulation in leaves of seven lines. Five out of these seven lines exhibited a reduction of StubGAL83 mRNA levels also in root and tuber tissues. Independent on the type of repression, the transgenic lines showed a delay in rooting and an increased sensitivity to salt stress. The roots were stunted and possessed less pronounced tap roots than the controls albeit with different severity in the different transgenic lines. The root cells were smaller and some of them had irregular shape. Tuberisation of the antisense-StubGAL83 lines was delayed, the size of the tubers was reduced while the number of tubers per plant was increased. These results together suggest that StubGAL83 affects root and tuber development probably by altering the metabolic status of the leaves.