Judith L. Roe

A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants.

Geminiviruses replicate in nuclei of mature plant cells after inducing the accumulation of host DNA replication machinery. Earlier studies showed that the viral replication factor, AL1, is sufficient for host induction and interacts with the cell cycle regulator, retinoblastoma (pRb). Unlike other DNA virus proteins, AL1 does not contain the pRb binding consensus, LXCXE, and interacts with plant pRb homo logues (pRBR) through a novel amino acid sequence. We mapped the pRBR binding domain of AL1 between amino acids 101 and 180 and identified two mutants that are differentially impacted for AL1–pRBR interactions. Plants infected with the E‐N140 mutant, which is wild‐type for pRBR binding, developed wild‐type symptoms and accumulated viral DNA and AL1 protein in epidermal, mesophyll and vascular cells of mature leaves. Plants inoculated with the KEE146 mutant, which retains 16% pRBR binding activity, only developed chlorosis along the veins, and viral DNA, AL1 protein and the host DNA synthesis factor, proliferating cell nuclear antigen, were localized to vascular tissue. These results established the importance of AL1–pRBR interactions during geminivirus infection of plants.

The Tousled gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development

Mutation at the TOUSLED locus of A. thaliana results in a complex phenotype, the most dramatic aspect of which being the abnormal flowers produced in mutant plants. tsl flowers show a random loss of floral organs, and organ development is impaired. The TSL gene appears to be required in the floral meristem for correct initiation of floral organ primordia and for proper development of organ primordia. Loss of TSL function also affects flowering time and leaf morphology. Using a mutation derived by T-DNA insertion mutagenesis, we have cloned the TSL gene and found that it encodes a protein kinase homolog with a novel N-terminal domain. This protein kinase gene identifies a novel signaling/regulatory pathway used during development in Arabidopsis.

Candidate Gene Association Mapping of Arabidopsis Flowering Time

The pathways responsible for flowering time in Arabidopsis thaliana comprise one of the best characterized genetic networks in plants. We harness this extensive molecular genetic knowledge to identify potential flowering time quantitative trait genes (QTGs) through candidate gene association mapping using 51 flowering time loci. We genotyped common single nucleotide polymorphisms (SNPs) at these genes in 275 A. thaliana accessions that were also phenotyped for flowering time and rosette leaf number in long and short days. Using structured association techniques, we find that haplotype-tagging SNPs in 27 flowering time genes show significant associations in various trait/environment combinations. After correction for multiple testing, between 2 and 10 genes remain significantly associated with flowering time, with CO arguably possessing the most promising associations. We also genotyped a subset of these flowering time gene SNPs in an independent recombinant inbred line population derived from the intercrossing of 19 accessions. Approximately one-third of significant polymorphisms that were associated with flowering time in the accessions and genotyped in the outbred population were replicated in both mapping populations, including SNPs at the CO, FLC, VIN3, PHYD, and GA1 loci, and coding region deletions at the FRI gene. We conservatively estimate that ∼4–14% of known flowering time genes may harbor common alleles that contribute to natural variation in this life history trait.

TOUSLED participates in apical tissue formation during gynoecium development in Arabidopsis.

Mutations at the TOUSLED (TSL) protein kinase locus in Arabidopsis cause reduced differentiation of apical gynoecial tissues and eliminate the fusion of the style and septum. TSL expression becomes confined to the developing style by stage 13, where it may promote expansion of tissues. Double mutant analysis suggests that ETTIN interacts with TSL, possibly by restricting TSL expression to apical regions. TSL, LEUNIG, and PERIANTHIA appear to participate in pathways of redundant function during the development of specific gynoecial tissues. TSL and LEUNIG most likely function in similar pathways during ovule development. TSL acts independently of the function of the organ identity genes AGAMOUS and APETALA2, and it is required for the formation of specific tissues in ectopic carpels. Mutations in TSL, ETTIN, PERIANTHIA, and LEUNIG all affect floral organ number as well as gynoecium morphology. Their respective wild-type loci must therefore play important roles in early floral meristem development during initiation of organ primordia in addition to their functions during regional differentiation within developing gynoecial primordia.

A Multiobjective Evolutionary-Simplex Hybrid Approach for the Optimization of Differential Equation Models of Gene Networks

This paper describes genetic and hybrid approaches for multiobjective optimization using a numerical measure called fuzzy dominance. Fuzzy dominance is used when implementing tournament selection within the genetic algorithm (GA). In the hybrid version, it is also used to carry out a Nelder-Mead simplex-based local search. The proposed GA is shown to perform better than NSGA-II and SPEA-2 on standard benchmarks, as well as for the optimization of a genetic model for flowering time control in rice. Adding the local search achieves faster convergence, an important feature in computationally intensive optimization of gene networks. The hybrid version also compares well with ParEGO on a few other benchmarks. The proposed hybrid algorithm is then applied to estimate the parameters of an elaborate gene network model of flowering time control in Arabidopsis. Overall solution quality is quite good by biological standards. Tradeoffs are discussed between accuracy in gene activity levels versus in the plant traits that they influence. These tradeoffs suggest that data mining the Pareto front may be useful in bioinformatics.

The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis

The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity.

TOUSLED Kinase Activity Oscillates during the Cell Cycle and Interacts with Chromatin Regulators

The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism.

Flowering time control: gene network modelling and the link to quantitative genetics

Flowering is a key stage in plant development that initiates grain production and is vulnerable to stress. The genes controlling flowering time in the model plant Arabidopsis thaliana are reviewed. Interactions between these genes have been described previously by qualitative network diagrams. We mathematically relate environmentally dependent transcription, RNA processing, translation, and protein–protein interaction rates to resultant phenotypes. We have developed models (reported elsewhere) based on these concepts that simulate flowering times for novel A. thaliana genotype–environment combinations. Here we draw 12 contrasts between genetic network (GN) models of this type and quantitative genetics (QG), showing that both have equal contributions to make to an ideal theory. Physiological dominance and additivity are examined as emergent properties in the context of feed-forwards networks, an instance of which is the signal-integration portion of the A. thaliana flowering time network. Additivity is seen to be a complex, multi-gene property with contributions from mass balance in transcript production, the feed-forwards structure itself, and downstream promoter reaction thermodynamics. Higher level emergent properties are exemplified by critical short daylength (CSDL), which we relate to gene expression dynamics in rice (Oryza sativa). Next to be discussed are synergies between QG and GN relating to the quantitative trait locus (QTL) mapping of model coefficients. This suggests a new verification test useful in GN model development and in identifying needed updates to existing crop models. Finally, the utility of simple models is evinced by 80 years of QG theory and mathematical ecology.

SKS6, a multicopper oxidase-like gene, participates in cotyledon vascular patterning during Arabidopsis thaliana development

SKU5-Similar 6 (SKS6) is a one of a large gene family of 19 members in Arabidopsis thaliana (L.) Heynh that encode multicopper oxidase-like proteins that are related to ferroxidases, ascorbate oxidases and laccases. Only one member of the family has been previously studied; Skewed5 (SKU5) is involved in the control of root growth. The encoded SKS6 protein, like SKU5 appears to lack a functional copper-binding site and is most closely related to Bp10 from Brassica napus and Ntp303 from Nicotiana tobacum. The SKS6 promoter contains many putative regulatory sites and differential expression of an SKS6::GUS reporter gene revealed selective induction in several seedling tissues including guard cells, root cortex cells, and leaf margin hydathodes. It was also expressed later in flower development in flower primordia, ovules, and the abscission zones of seeds and siliques. Furthermore, SKS6 was upregulated in roots in response to treatment of seedlings with the hormones abscisic acid, indole-3 acetic acid, 2,4-dichlorophenoxyacetic acid and aminocyclopropane-1-carboxylate. A loss-of function sks6-1 T-DNA insertion allele revealed that cotyledon vascular patterning is affected in the mutant, suggesting a role for the protein in metabolism of nutrients or hormones in the hydathodes, the sites of auxin synthesis and chemical recycling.

Fuzzy Dominance Based Multi-objective GA-Simplex Hybrid Algorithms Applied to Gene Network Models

Hybrid algorithms that combine genetic algorithms with the Nelder-Mead simplex algorithm have been effective in solving certain optimization problems. In this article, we apply a similar technique to estimate the parameters of a gene regulatory network for flowering time control in rice. The algorithm minimizes the difference between the model behavior and real world data. Because of the nature of the data, a multi-objective approach is necessary. The concept of fuzzy dominance is introduced, and a multi-objective simplex algorithm based on this concept is proposed as a part of the hybrid approach. Results suggest that the proposed method performs well in estimating the model parameters.