The control of flowering time in plants is critical for plant fitness and for agriculture. The genetic pathways governing this developmental transition are reasonably well understood in Arabidopsis, although substantial new gains are still being made in this system. Much new work is focusing on how the genetic networks governing flowering function in other species.

Background

Tomato species are of significant agricultural and ecological interest, with cultivated tomato being among the most common vegetable crops grown. Wild tomato species are native to diverse habitats in South America and show great morphological and ecological diversity that has proven useful in breeding programs. However, relatively little is known about nucleotide diversity between tomato species. Until recently limited sequence information was available for tomato, preventing genome-wide evolutionary analyses. Now, an extensive collection of tomato expressed sequence tags (ESTs) is available at the SOL Genomics Network (SGN). This database holds sequences from several species, annotated with quality values, assembled into unigenes, and tested for homology against other genomes. Despite the importance of polymorphism detection for breeding and natural variation studies, such analyses in tomato have mostly been restricted to cultivated accessions. Importantly, previous polymorphisms surveys mostly ignored the linked meta-information, limiting functional and evolutionary analyses. The current data in SGN is thus an under-exploited resource. Here we describe a cross-species analysis taking full-advantage of available information.

Results

We mined 20,000 interspecific polymorphisms between Solanum lycopersicum and S. habrochaites or S. pennellii and 28,800 intraspecific polymorphisms within S. lycopersicum. Using the available meta-information we classified genes into functional categories and obtained estimations of single nucleotide polymorphisms (SNP) quality, position in the gene, and effect on the encoded proteins, allowing us to perform evolutionary analyses. Finally, we developed a set of more than 10,000 between-species molecular markers optimized by sequence quality and predicted intron position. Experimental validation of 491 of these molecular markers resulted in confirmation of 413 polymorphisms.

Conclusion

We present a new analysis of the extensive tomato EST sequences available that represents the most comprehensive survey of sequence diversity across Solanum species to date. These SNPs, plus thousands of molecular makers designed to detect the polymorphisms are available to the community via a website. Evolutionary analyses on these polymorphism uncovered sets of genes potentially important for the evolution and domestication of tomato; interestingly these sets were enriched for genes involved in response to the environment.

Plants have a variety of strategies to avoid canopy shade and compete with their neighbors for light, collectively called the shade avoidance syndrome (SAS). Plants also have extensive systems to defend themselves against pathogens and herbivores. Defense and shade avoidance are two fundamental components of plant survival and productivity, and there are often tradeoffs between growth and defense. Recently, MYC2, a major positive regulator of defense, was reported to inhibit elongation during shade avoidance. Here, we further investigate the role of MYC2 and the related MYC3 and MYC4 in shade avoidance, and we examine the relationship between MYC2/3/4 and the PIF family of light-regulated transcription factors. We demonstrate that MYC2/3/4 inhibit both elongation and flowering. Furthermore, using both genetic and transcriptomic analysis we find that MYCs and PIFs generally function independently in growth regulation. However, surprisingly, the pif4/5/7 triple mutant restored the petiole shade avoidance response of myc2 (jin1-2) and myc2/3/4 We theorize that increased petiole elongation in myc2/3/4 could be more due to resource tradeoffs or post-translational modifications rather than interactions with PIF4/5/7 affecting gene regulation.

Several closely related Myb-like activator proteins are known to have partially redundant functions within the plant circadian clock, but their specific roles are not well understood. To clarify the function of the REVEILLE 4, REVEILLE 6, and REVEILLE 8 transcriptional activators, we characterized the growth and clock phenotypes of CRISPR-Cas9-generated single, double, and triple rve mutants. We found that these genes act synergistically to regulate flowering time, redundantly to regulate leaf growth, and antagonistically to regulate hypocotyl elongation. We previously reported that increasing intensities of monochromatic blue and red light have opposite effects on the period of triple rve468 mutants. Here, we further examined light quality-specific phenotypes of rve mutants and report that rve468 mutants lack the blue light-specific increase in expression of some circadian clock genes observed in wild type. To investigate the basis of these blue light-specific circadian phenotypes, we examined RVE protein abundances and degradation rates in blue and red light and found no significant differences between these conditions. We next examined genetic interactions between RVE genes and ZEITLUPE and ELONGATED HYPOCOTYL5, two factors with blue light-specific functions in the clock. We found that the RVEs interact additively with both ZEITLUPE and ELONGATED HYPOCOTYL5 to regulate circadian period, which suggests that neither of these factors are required for the blue light-specific differences that we observed. Overall, our results suggest that the RVEs have separable functions in plant growth and circadian regulation and that they are involved in blue light-specific circadian signaling via a novel mechanism.

The phytochrome (phy) family of red and far-red photoreceptors provides plants with critical information about their surrounding environment and can signal downstream developmental and physiological changes. Neighboring plants compete for limited light resources, and their presence is detected by the phytochrome photoreceptors as a reduced ratio of red: far-red light. One common response to shade is increased elongation of petioles and internodes to compete with their neighbors. While the phytochrome family, phyB in particular, has been well studied in Arabidopsis, information about the other phytochrome family members is limited, especially in sympodial crop plants such as tomato, that have a very different architecture from that of the model plant. To study the tomato phytochrome family we took advantage of several existing mutants and generated an artificial miRNA (amiRNA) line to target SlPHYE, the remaining phytochrome B subfamily member with no currently available mutant line. Here, we characterize internode elongation and shade avoidance phenotypes of the SlPHYE amiRNA line (PHYE amiRNA). In addition, higher order phytochrome subfamily B mutants were generated with the PHYE amiRNA line to investigate the role of SlphyE within the phyB subfamily. We find that the PHYE amiRNA line has no detectable phenotype on its own, however in higher order combinations with SlphyB1 and/or SlphyB2 there are notable defects in shade avoidance. Most notably, we find that the triple mutant combination of SlPHYE amiRNA, SlphyB1, and SlphyB2 has a phenotype that is much stronger than the SlphyB1 SlphyB2 double, showing constitutive shade avoidance and little to no response to shade. This indicates that SlphyE is required for the shade avoidance response in the absence of SlphyB1 and SlphyB2.

Shade avoidance is an ecologically and molecularly well-understood set of plant developmental responses that occur when the ratio of red to far-red light (R:FR) is reduced as a result of foliar shade. Here, a genome-wide association study (GWAS) in Arabidopsis thaliana was used to identify variants underlying one of these responses: increased hypocotyl elongation. Four hypocotyl phenotypes were included in the study, including height in high R:FR conditions (simulated sun), height in low R:FR conditions (simulated shade), and two different indices of the response of height to low R:FR. GWAS results showed that variation in these traits is controlled by many loci of small to moderate effect. A known PHYC variant contributing to hypocotyl height variation was identified and lists of significantly associated genes were enriched in a priori candidates, suggesting that this GWAS was capable of generating meaningful results. Using metadata such as expression data, GO terms, and other annotation, we were also able to identify variants in candidate de novo genes. Patterns of significance among our four phenotypes allowed us to categorize associations into three groups: those that affected hypocotyl height without influencing shade avoidance, those that affected shade avoidance in a height-dependent fashion, and those that exerted specific control over shade avoidance. This grouping allowed for the development of explicit hypotheses about the genetics underlying shade avoidance variation. Additionally, the response to shade did not exhibit any marked geographic distribution, suggesting that variation in low R:FR-induced hypocotyl elongation may represent a response to local conditions.

Background

As 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 Earth's 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.

Results

We 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.

Conclusion

Our 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.

Quantitative genetic analysis has long been used to study how natural variation of genotype can influence an organism's phenotype. While most studies have focused on genetic determinants of phenotypic average, it is rapidly becoming understood that stochastic noise is genetically determined. However, it is not known how many traits display genetic control of stochastic noise nor how broadly these stochastic loci are distributed within the genome. Understanding these questions is critical to our understanding of quantitative traits and how they relate to the underlying causal loci, especially since stochastic noise may be directly influenced by underlying changes in the wiring of regulatory networks. We identified QTLs controlling natural variation in stochastic noise of glucosinolates, plant defense metabolites, as well as QTLs for stochastic noise of related transcripts. These loci included stochastic noise QTLs unique for either transcript or metabolite variation. Validation of these loci showed that genetic polymorphism within the regulatory network alters stochastic noise independent of effects on corresponding average levels. We examined this phenomenon more globally, using transcriptomic datasets, and found that the Arabidopsis transcriptome exhibits significant, heritable differences in stochastic noise. Further analysis allowed us to identify QTLs that control genomic stochastic noise. Some genomic QTL were in common with those altering average transcript abundance, while others were unique to stochastic noise. Using a single isogenic population, we confirmed that natural variation at ELF3 alters stochastic noise in the circadian clock and metabolism. Since polymorphisms controlling stochastic noise in genomic phenotypes exist within wild germplasm for naturally selected phenotypes, this suggests that analysis of Arabidopsis evolution should account for genetic control of stochastic variance and average phenotypes. It remains to be determined if natural genetic variation controlling stochasticity is equally distributed across the genomes of other multi-cellular eukaryotes.