Julin N. Maloof

Rhythmic growth explained by coincidence between internal and external cues

Most organisms use circadian oscillators to coordinate physiological and developmental processes such as growth with predictable daily environmental changes like sunrise and sunset. The importance of such coordination is highlighted by studies showing that circadian dysfunction causes reduced fitness in bacteria and plants, as well as sleep and psychological disorders in humans. Plant cell growth requires energy and water—factors that oscillate owing to diurnal environmental changes. Indeed, two important factors controlling stem growth are the internal circadian oscillator and external light levels. However, most circadian studies have been performed in constant conditions, precluding mechanistic study of interactions between the clock and diurnal variation in the environment. Studies of stem elongation in diurnal conditions have revealed complex growth patterns, but no mechanism has been described. Here we show that the growth phase of Arabidopsis seedlings in diurnal light conditions is shifted 8–12 h relative to plants in continuous light, and we describe a mechanism underlying this environmental response. We find that the clock regulates transcript levels of two basic helix–loop–helix genes, phytochrome-interacting factor 4 (PIF4) and PIF5, whereas light regulates their protein abundance. These genes function as positive growth regulators; the coincidence of high transcript levels (by the clock) and protein accumulation (in the dark) allows them to promote plant growth at the end of the night. Thus, these two genes integrate clock and light signalling, and their coordinated regulation explains the observed diurnal growth rhythms. This interaction may serve as a paradigm for understanding how endogenous and environmental signals cooperate to control other processes.

Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development

BackgroundAs nonmotile organisms, plants must rapidly adapt to ever-changing environmental conditions, including those caused by daily light/dark cycles. One important mechanism for anticipating and preparing for such predictable changes is the circadian clock. Nearly all organisms have circadian oscillators that, when they are in phase with the Earths rotation, provide a competitive advantage. In order to understand how circadian clocks benefit plants, it is necessary to identify the pathways and processes that are clock controlled.ResultsWe have integrated information from multiple circadian microarray experiments performed on Arabidopsis thaliana in order to better estimate the fraction of the plant transcriptome that is circadian regulated. Analyzing the promoters of clock-controlled genes, we identified circadian clock regulatory elements correlated with phase-specific transcript accumulation. We have also identified several physiological pathways enriched for clock-regulated changes in transcript abundance, suggesting they may be modulated by the circadian clock.ConclusionOur analysis suggests that transcript abundance of roughly one-third of expressed A. thaliana genes is circadian regulated. We found four promoter elements, enriched in the promoters of genes with four discrete phases, which may contribute to the time-of-day specific changes in the transcript abundance of these genes. Clock-regulated genes are over-represented among all of the classical plant hormone and multiple stress response pathways, suggesting that all of these pathways are influenced by the circadian clock. Further exploration of the links between the clock and these pathways will lead to a better understanding of how the circadian clock affects plant growth and leads to improved fitness.

The extent of linkage disequilibrium in Arabidopsis thaliana

Linkage disequilibrium (LD), the nonrandom occurrence of alleles in haplotypes, has long been of interest to population geneticists. Recently, the rapidly increasing availability of genomic polymorphism data has fueled interest in LD as a tool for fine-scale mapping, in particular for human disease loci. The chromosomal extent of LD is crucial in this context, because it determines how dense a map must be for associations to be detected and, conversely, limits how finely loci may be mapped. Arabidopsis thaliana is expected to harbor unusually extensive LD because of its high degree of selfing. Several polymorphism studies have found very strong LD within individual loci, but also evidence of some recombination. Here we investigate the pattern of LD on a genomic scale and show that in global samples, LD decays within approximately 1 cM, or 250 kb. We also show that LD in local populations may be much stronger than that of global populations, presumably as a result of founder events. The combination of a relatively high level of polymorphism and extensive haplotype structure bodes well for developing a genome-wide LD map in A. thaliana.

Natural variation in light sensitivity of Arabidopsis.

Because plants depend on light for growth, their development and physiology must suit the particular light environment. Plants native to different environments show heritable, apparently adaptive, changes in their response to light. As a first step in unraveling the genetic and molecular basis of these naturally occurring differences, we have characterized intraspecific variation in a light-dependent developmental process—seedling emergence. We examined 141 Arabidopsis thaliana accessions for their response to four light conditions, two hormone conditions and darkness. There was significant variation in all conditions, confirming that Arabidopsis is a rich source of natural genetic diversity. Hierarchical clustering revealed that some accessions had response patterns similar to known photoreceptor mutants, suggesting changes in specific signaling pathways. We found that the unusual far-red response of the Lm-2 accession is due to a single amino-acid change in the phytochrome A (PHYA) protein. This change stabilizes the light-labile PHYA protein in light and causes a 100-fold shift in the threshold for far-red light sensitivity. Purified recombinant Lm-2 PHYA also shows subtle photochemical differences and has a reduced capacity for autophosphorylation. These biochemical changes contrast with previously characterized natural alleles in loci controlling plant development, which result in altered gene expression or loss of gene function.

The PHYTOCHROME C photoreceptor gene mediates natural variation in flowering and growth responses of Arabidopsis thaliana.

Light has an important role in modulating seedling growth and flowering time. We show that allelic variation at the PHYTOCHROME C (PHYC) photoreceptor locus affects both traits in natural populations of A. thaliana. Two functionally distinct PHYC haplotype groups are distributed in a latitudinal cline dependent on FRIGIDA, a locus that together with FLOWERING LOCUS C explains a large portion of the variation in A. thaliana flowering time. In a genome-wide scan for association of 65 loci with latitude, there was an excess of significant P values, indicative of population structure. Nevertheless, PHYC was the most strongly associated locus across 163 strains, suggesting that PHYC alleles are under diversifying selection in A. thaliana. Our work, together with previous findings, suggests that photoreceptor genes are major agents of natural variation in plant flowering and growth response.

Comparative transcriptomics reveals patterns of selection in domesticated and wild tomato

Significance One of the most important technological advances by humans is the domestication of plant species for the production of food. We have used high-throughput sequencing to identify changes in DNA sequence and gene expression that differentiate cultivated tomato and its wild relatives. We also identify hundreds of candidate genes that have evolved new protein sequences or have changed expression levels in response to natural selection in wild tomato relatives. Taken together, our analyses provide a snapshot of genome evolution under artificial and natural conditions. Although applied over extremely short timescales, artificial selection has dramatically altered the form, physiology, and life history of cultivated plants. We have used RNAseq to define both gene sequence and expression divergence between cultivated tomato and five related wild species. Based on sequence differences, we detect footprints of positive selection in over 50 genes. We also document thousands of shifts in gene-expression level, many of which resulted from changes in selection pressure. These rapidly evolving genes are commonly associated with environmental response and stress tolerance. The importance of environmental inputs during evolution of gene expression is further highlighted by large-scale alteration of the light response coexpression network between wild and cultivated accessions. Human manipulation of the genome has heavily impacted the tomato transcriptome through directed admixture and by indirectly favoring nonsynonymous over synonymous substitutions. Taken together, our results shed light on the pervasive effects artificial and natural selection have had on the transcriptomes of tomato and its wild relatives.

The genome of the stress-tolerant wild tomato species Solanum pennellii

Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm.

Genomic Analysis of Circadian Clock-, Light-, and Growth-Correlated Genes Reveals PHYTOCHROME-INTERACTING FACTOR5 as a Modulator of Auxin Signaling in Arabidopsis

Plants exhibit daily rhythms in their growth, providing an ideal system for the study of interactions between environmental stimuli such as light and internal regulators such as the circadian clock. We previously found that two basic loop-helix-loop transcription factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, integrate light and circadian clock signaling to generate rhythmic plant growth in Arabidopsis (Arabidopsis thaliana). Here, we use expression profiling and real-time growth assays to identify growth regulatory networks downstream of PIF4 and PIF5. Genome-wide analysis of light-, clock-, or growth-correlated genes showed significant overlap between the transcriptomes of clock-, light-, and growth-related pathways. Overrepresentation analysis of growth-correlated genes predicted that the auxin and gibberellic acid (GA) hormone pathways both contribute to diurnal growth control. Indeed, lesions of GA biosynthesis genes retarded rhythmic growth. Surprisingly, GA-responsive genes are not enriched among genes regulated by PIF4 and PIF5, whereas auxin pathway and response genes are. Consistent with this finding, the auxin response is more severely affected than the GA response in pif4 pif5 double mutants and in PIF5-overexpressing lines. We conclude that at least two downstream modules participate in diurnal rhythmic hypocotyl growth: PIF4 and/or PIF5 modulation of auxin-related pathways and PIF-independent regulation of the GA pathway.

Network Quantitative Trait Loci Mapping of Circadian Clock Outputs Identifies Metabolic Pathway-to-Clock Linkages in Arabidopsis

The authors developed a network approach that simplifies the identification of natural variation in complex temporal systems. Using this approach, they identified significant linkages between natural variation in the circadian clock and plant metabolism, including a newly described capacity for structural variation in plant secondary metabolism to influence the periodicity of the circadian clock. Modern systems biology permits the study of complex networks, such as circadian clocks, and the use of complex methodologies, such as quantitative genetics. However, it is difficult to combine these approaches due to factorial expansion in experiments when networks are examined using complex methods. We developed a genomic quantitative genetic approach to overcome this problem, allowing us to examine the function(s) of the plant circadian clock in different populations derived from natural accessions. Using existing microarray data, we defined 24 circadian time phase groups (i.e., groups of genes with peak phases of expression at particular times of day). These groups were used to examine natural variation in circadian clock function using existing single time point microarray experiments from a recombinant inbred line population. We identified naturally variable loci that altered circadian clock outputs and linked these circadian quantitative trait loci to preexisting metabolomics quantitative trait loci, thereby identifying possible links between clock function and metabolism. Using single-gene isogenic lines, we found that circadian clock output was altered by natural variation in Arabidopsis thaliana secondary metabolism. Specifically, genetic manipulation of a secondary metabolic enzyme led to altered free-running rhythms. This represents a unique and valuable approach to the study of complex networks using quantitative genetics.

A Quantitative Genetic Basis for Leaf Morphology in a Set of Precisely Defined Tomato Introgression Lines

Natural variation leading to differences in leaf morphology between domesticated tomato and a wild relative is explored in a set of introgression lines. The phenotypic context of leaf morphology with other traits is examined at the whole-plant level, with implications for organ-specific breeding efforts. Introgression lines (ILs), in which genetic material from wild tomato species is introgressed into a domesticated background, have been used extensively in tomato (Solanum lycopersicum) improvement. Here, we genotype an IL population derived from the wild desert tomato Solanum pennellii at ultrahigh density, providing the exact gene content harbored by each line. To take advantage of this information, we determine IL phenotypes for a suite of vegetative traits, ranging from leaf complexity, shape, and size to cellular traits, such as stomatal density and epidermal cell phenotypes. Elliptical Fourier descriptors on leaflet outlines provide a global analysis of highly heritable, intricate aspects of leaf morphology. We also demonstrate constraints between leaflet size and leaf complexity, pavement cell size, and stomatal density and show independent segregation of traits previously assumed to be genetically coregulated. Meta-analysis of previously measured traits in the ILs shows an unexpected relationship between leaf morphology and fruit sugar levels, which RNA-Seq data suggest may be attributable to genetically coregulated changes in fruit morphology or the impact of leaf shape on photosynthesis. Together, our results both improve upon the utility of an important genetic resource and attest to a complex, genetic basis for differences in leaf morphology between natural populations.