Plants exhibit different physiological and molecular responses to adverse changes in their environment. One such molecular response is the sequestration of proteins, RNAs, and metabolites into cytoplasmic bodies called stress granules (cSGs). Here we report that, in addition to cSGs, heat stress also induces the formation of SG-like foci (cGs) in the chloroplasts of the model plant Arabidopsis thaliana . Similarly to the cSGs, (i) cpSG assemble rapidly in response to stress and disappear when the stress ceases, (ii) cpSG formation is inhibited by treatment with a translation inhibitor (lincomycin), and (iii) cpSG are composed of a stable core and a fluid outer shell. A previously published protocol for cSG extraction was successfully adapted to isolate cpSG, followed by protein, metabolite, and RNA analysis. Analogously to the cSGs, cpSG sequester proteins essential for SG formation, dynamics, and function, also including RNA-binding proteins with prion-like domain, ATPases and chaperones, and the amino acids proline and glutamic acid. However, the most intriguing observation relates to the cpSG localization of proteins, such as a complete magnesium chelatase complex, which is involved in photosynthetic acclimation to stress. These data suggest that cpSG have a role in plant stress tolerance.

26The biotic and the abiotic environment play a major role in shaping plant 27 phenotypes and their geographic distributions. However, little is known about the 28 extent to which plant phenotypes match local patterns of herbivory across fine-29 grained habitat mosaics, despite the strong effect of herbivory on plant fitness. 30 Through a reciprocal transplant-common garden experiment with clonally 31 propagated rhizomes, we tested for local phenotypic differentiation in bittercress 32 (Brassicaceae: Cardamine cordifolia) plants collected across an ecotonal habitat 33 mosaic. We found that bittercress in sunny meadows (high herbivory) and shaded 34 understories (low herbivory) have diverged in heritable growth and herbivore 35 resistance phenotypes. The expression of these differences was habitat dependent, 36 mirroring patterns of adaptive divergence in phenotypic plasticity between plant 37 populations in meadow and understory habitats at broader geographic scales, and 38 showed no evidence for a constraint imposed by growth-defense tradeoffs. Most 39 notably, plants derived from shade habitats exhibited a weaker shade-induced 40 elongation response (i.e., shade avoidance syndrome, SAS) and reduced resistance 41 to herbivory, relative to plants derived from sun habitats, when both were grown in 42 shade common gardens. Greenhouse experiments revealed that divergent SAS 43 phenotypes in shade conditions were expressed in offspring grown from seed as well. 44Finally, we observed partially non-overlapping flowering phenology between 45 habitat-types in the field, which may be at least one factor that helps to reinforce 46 habitat-specific phenotypic divergence. Altogether, our study illuminates how a 47 48 grained habitat mosaic. 49 50 51 55 2005; Richardson et al. 2014). However, our knowledge regarding the conditions 56 under which these patterns arise and persist has been disproportionately shaped by 57 studies at course-grained rather than fine-grained spatial scales (Richardson et al.58 2014). This bias has likely been shaped by the expectation that gene flow among 59 interspersed habitat patches is generally a strong homogenizing force, preventing 60 the establishment of habitat-associated phenotypic and genotypic variation at fine-61 grained spatial scales (Haldane 1930; Lenormand 2002). Although there is growing 62 evidence that heritable phenotypes track habitat mosaics at fine-grained spatial 63 3 scales [i.e. microgeographic phenotypic divergence; (Richardson et al. 2014)] we 64 have a limited understanding of the molecular, ecological, and evolutionary 65 processes that facilitate and maintain this variation in nature. 66 67 Defoliation by insect herbivores exerts strong selection on plant phenotypes (Louda 68 1984; Prasad et al. 2012; Agrawal et al. 2012). In mustards (Brassicaceae), 69 polymorphisms in genes that modify defensive chemicals underlie adaptation to 70 local herbivore communities (Prasad et al. 2012; Zust et al. 2012), and the 71 magnitude of geographic divergence at such loci is extr...

Organismal phenotypes often co-vary with environmental variables across broad geographic ranges. Less is known about the extent to which phenotypes match local conditions when multiple biotic and abiotic stressors vary at fine spatial scales. Bittercress (Brassicaceae: Cardamine cordifolia), a perennial forb, grows across a microgeographic mosaic of two contrasting herbivory regimes: high herbivory in meadows (sun habitats) and low herbivory in deeply shaded forest understories (shade habitats). We tested for local phenotypic differentiation in plant size, leaf morphology, and anti-herbivore defense (realized resistance and defensive chemicals, i.e., glucosinolates) across this habitat mosaic through reciprocal transplant-common garden experiments with clonally propagated rhizomes. We found habitat-specific divergence in morphological and defensive phenotypes that manifested as contrasting responses to growth in shade common gardens: weak petiole elongation and attenuated defenses in populations from shade habitats, and strong petiole elongation and elevated defenses in populations from sun habitats. These divergent phenotypes are generally consistent with reciprocal local adaptation: plants from shade habitats that naturally experience low herbivory show reduced investment in defense and an attenuated shade avoidance response, owing to its ineffectiveness within forest understories. By contrast, plants from sun habitats with high herbivory show shade-induced elongation, but no evidence of attenuated defenses canonically associated with elongation in shade-intolerant plant species. Finally, we observed differences in flowering phenology between habitat types that could potentially contribute to inter-habitat divergence by reducing gene flow. This study illuminates how clonally heritable plant phenotypes track a fine-grained mosaic of herbivore pressure and light availability in a native plant.

AbstractThe telomerase ribonucleoprotein complex (RNP) is essential for genome stability and performs this role through the addition of repetitive DNA to the ends of chromosomes. The telomerase enzyme is composed of a reverse transcriptase (TERT), which utilizes a template domain in an RNA subunit (TER) to reiteratively add telomeric DNA at the ends of chromosomes. Multiple TERs have been identified in the model plant Arabidopsis thaliana. Here we combine a phylogenetic and biochemical approach to understand how the telomerase RNP has evolved in Brassicaceae, the family that includes A. thaliana. Because of the complex phylogenetic pattern of template domain loss and alteration at the previously characterized A. thaliana TER loci, TER1 and TER2, across the plant family Brassicaceae, we bred double mutants from plants with a template deletion at AtTER1 and T-DNA insertion at AtTER2. These double mutants exhibited no telomere length deficiency, a definitive indication that neither of these loci encode a functional telomerase RNA. Moreover, we determined that the telomerase components TERT, Dyskerin, and the KU heterodimer are under strong purifying selection, consistent with the idea that the TER with which they interact is also conserved. To test this hypothesis further, we analyzed the substrate specificity of telomerase from species across Brassicaceae and determined that telomerase from close relatives bind and extend substrates in a similar manner, supporting the idea that TERs in different species are highly similar to one another and are likely encoded from an orthologous locus. Lastly, TERT proteins from across Brassicaceae were able to complement loss of function tert mutants in vivo, indicating TERTs from other species have the ability to recognize the native TER of A. thaliana. Finally, we immunoprecipitated the telomerase complex and identified associated RNAs via RNA-seq. Using our evolutionary data we constrained our analyses to conserved RNAs within Brassicaceae that contained a template domain. These analyses revealed a highly expressed locus whose disruption by a T-DNA resulted in a telomeric phenotype similar to the loss of other telomerase core proteins, indicating that the RNA has an important function in telomere maintenance.