It remains to be seen whether AM groups might vary as well.Several studies have detailed changes caused by N deposition or fertilization in the species assemblage of mushrooms . However, as the community composition of ECM fruiting bodies can differ from that of below-ground ECM structures , and below-ground biomass could be a substantial C pool, this review focuses on the species composition of fungal structures in the soil. In both a Swedish Picea abies forest and a Scottish Sitka spruce plantation , frequencies of ECM morphotypes on root tips shifted upon long-term N fertilization. Likewise, in Pinus sylvestris forests in Sweden, colonization of ‘bait’ seedlings by one particular morphotype was less frequent in N fertilized than in control plots . Composition of AM communities can also shift with N availability. In a natural N deposition gradient in southern California coastal sage scrub, spores of Scutellospora and Gigaspora species became less prevalent with increasing deposition. Conversely, spores of certain Glomus species proliferated under the same conditions . In addition, in tall grass prairie the abundance of spores from Gigaspora gigantea and Glomus mosseae increased with N fertilization, while that of Entrophospora infrequens declined significantly . Johnson noted an increase in the presence of Gigaspora gigantea, Gigaspora margarita, Scutellospora calospora and Glomus occultum, and a decrease in Glomus intraradix after 8 yr fertilization with a suite of nutrients including N and P. Alterations in the species assemblage of mycorrhizal fungi, either directly through N availability or indirectly through shifts in plant communities,dutch bucket for tomatoes appear to be a likely outcome of widespread N deposition.

Nitrogen effects on the survivorship and decomposition of mycorrhizal hyphae have received little attention. Using minirhizotrons, Majdi & Nylund found that N fertilization significantly reduced the life span of ectomycorrhizal short roots from 240 to 210 d in a 30-yr-old Norway spruce stand in southwest Sweden. The authors did not speculate on mechanisms underpinning this change, but we suggest that alterations of the ECM community could have been one factor. In another minirhizotron experiment, Rygiewicz et al. recorded no effect of N fertilization on the length of time between appearance and disappearance of ECM tips. This latter result includes both life span and decomposition rate of the fungal tissue. These studies present valuable information on the dynamics of mycorrhizal root tips. However, alterations in turnover of extraradical ECM and AM hyphae by N additions have yet to be reported in the literature. Nitrogen concentrations of hyphae from one P. involutus isolate were reported to increase with higher N availability in a culture experiment, with possible shifts in decomposition rate. Additionally, root turnover can increase with N fertilization , and this response could produce a corresponding decrease in the life span of ECM structures such as mycorrhizal root tips and rhizomorphs.Increases in N availability inconsistently affect hyphal dynamics . In field studies, this variation in response could be partially attributable to the initial N status of the ecosystem; N-limited systems might respond differently from systems in which another factor limits primary productivity. Nutrient limitation of plants is an important consideration because mycorrhizal fungi derive the majority of their C from the host. However, this possibility has not been explicitly tested. At this point we can only suggest that C storage in living and recently dead mycorrhizal tissue might only be affected in certain systems exposed to N deposition. Furthermore, in glasshouse or culture studies the use of different fungal species or isolates might contribute to conflicting responses of hyphal growth or biomass. Even isolates of the same species can vary in response to N availability . Overall, alteration in the community composition of mycorrhizal fungi appears to be the most general response to N addition. Because mycorrhizal groups can vary in chitin content and growth rate, these shifts could have important consequences for C immobilization in live hyphal tissue or its residual soil organic matter.

However, a more complete understanding of differences in tissue quality and physiology among groups is necessary in order to predict their influence on C dynamics.Cell size correlates strongly with key aspects of cell physiology, including organelle abundance and DNA ploidy.While cells employ diverse strategies to regulate their size in different situations, it is unclear how these mechanisms are integrated to provide robust, systems-level control. In budding yeast, a molecular size sensor restricts passage of small cells through G1, enabling them to gain proportionally more volume than larger cells before progressing to Start. In contrast, size control post-Start is less clear. The duration of S/G2/M in wild type cells has been reported to exhibit only a weak dependence on cell size, so larger cells would be expected to add a greater volume than smaller ones. Yet it is also the case that even large mother cells produce smaller daughter cells, suggesting that additional regulation may play a role during S/G2/M, either by limiting bud growth rate or shortening the duration of budding. There is also conflicting evidence regarding the molecular size control mechanisms that might operate during S/G2/M, such as whether the kinase Swe1, the budding yeast homolog of fission yeast Wee1, regulates growth by sensing bud size or bud morphogenesis. Furthermore, while G1 size control mechanisms act on cells smaller than their set-point size, no mechanisms have been clearly defined to limit size as cells become larger. Since physiological perturbations can result in abnormally large cells, mechanisms must exist to ensure cells that grow too large are able to return to the set-point volume after successive rounds of growth and division. Underscoring the importance of this aspect of size homeostasis, tumor cells lacking functional size-homeostasis pathways often grow far larger than normal. In sum, how cells regulate size during S/G2/M and whether such mechanisms might enforce an upper limit on cell volume remains an important open question. To gain further insight into post-Start size control, we prepared ‘giant’ yeast using two approaches to reversibly block cell cycle progression but not growth: optogenetic disruption of the cell polarity factor Bem1 or a temperature sensitive cdk1 allele.

We reasoned that giant yeast would satisfy pre-Start size control while enabling us to uncover post-Start size-limiting mechanisms though the identification of invariant growth parameters . Upon release from their block, giant mothers reentered the cell cycle and populations of their progeny returned to their unperturbed size within hours. Volume regulation in these cells was inconsistent with two major classes of size control mechanisms: an ‘adder’ specifying a constant volume increment added over the course of a cell cycle and a ‘sizer’ specifying daughter cell volume. Instead,blueberry grow pot our data support a ‘timer’ mechanism that specifies the duration of S/G2/ M across the full range of daughter sizes. Our data thus provide evidence that cell size homeostasis is maintained by at least two separable mechanisms of size control: a pre-Start size sensor enabling size-dependent passage through Start, and a post-Start timer ensuring that daughters are smaller than their mothers. Together, these mechanisms ensure that yeast populations generated from cells at either size extreme rapidly return to a set-point within only a few cell division cycles.To achieve reversible control over cell size in the budding yeast S. cerevisiae, we first took advantage of the light-responsive PhyB/PIF optogenetic system to control the localization of Bem1, a cell polarity factor . In this “optoBem1” system, red light illumination relocalizes the PIF-Bem1 fusion protein to mitochondria-anchored PhyB . Light induced Bem1 re-localization produces an acute loss-of-function phenotype where cells fail to form a site of polarized Cdc42 activity, fail to initiate budding, and instead undergo continuous isotropic growth. Strikingly, this effect is quickly reversed upon illumination with infrared light, which releases PIF-Bem1 from the mitochondria within seconds. Upon release, cells form a bud within minutes and proceed to cytokinesis. The PIF-Bem1 fusion protein appears to fully recapitulate normal Bem1 function: when it is not sequestered to the mitochondria, overall cell sizes and cell growth rates are similar to an isogenic wild type strain. We performed additional experiments to more completely characterize optoBem1 giant cells. Our initial experiments quantifying the growth of red light-illuminated optoBem1 cells revealed two sub-populations of cells that grew at different rates . We hypothesized that cell growth rates differed depending on the cell cycle phase at the time of Bem1disruption. Indeed, we found that synchronizing optoBem1 cells before red light stimulation led to unimodally-distributed growth . Furthermore, restricting our analysis to measure growth only following entry into G1 yielded a unimodal distribution . We also observed that a substantial fraction of optoBem1 yeast burst as they become increasingly large , and hypothesized that cell lysis may be a result of large cells’ increased susceptibility to osmotic pressure. Supporting this hypothesis, growing cells in high-osmolarity media containing 1 M sorbitol decreased the frequency of cell lysis without affecting the rate of isotropic growth .

We therefore supplemented our media with sorbitol for all subsequent experiments involving optoBem1-arrested cells. Finally, to test whether growth was isotropic during the entire time period, we pulsed cells with fluorescent Concanavalin A to mark the existing cell wall, followed by a washout of free FITC-ConA. We found that cells exhibited uniform dilution of FITC-ConA around their surface, consistent with isotropic growth .Prior studies have established that unperturbed, freely-cycling budding yeast cells appear to exhibit an exponential growth in volume over time. However, most of this growth is localized to the bud, with only a minor contribution from the mother cell’s isotropic growth during G1. The mode of growth may also change depending on cell cycle phase. Since distinguishing between growth patterns is difficult to achieve during the growth interval of normal sized yeast, we reasoned that the ability to prepare isotropically-growing yeast with volumes spanning an order of magnitude would permit high-quality measurements of this growth law, and potentially reveal processes that limit cell growth as size increases.We imaged optoBem1 cells during red light illumination at multiple z-planes and used a custom code to automatically measure cell diameter every 10 min over a 12 h period. Following entry into G1 after Bem1 arrest, we found that isotropically-growing optoBem1 cells exhibited a linear increase in cell diameter over time, corresponding to a rate of volume growth proportional to 3 . Since these volume increases also show a strong correlation with protein content, as assessed by fluorescence, our data suggest that the growth we observed primarily arises from increases in cell mass rather than cell swelling . This result is inconsistent with two classic models of cell growth: a constant growth law, where volume increases linearly over time; and exponential growth, where the rate of growth is proportional to the cell’s current volume. In contrast, a linear increase in cell diameter is the expected result for volume increasing in proportion to cell surface area . Surface area-proportional growth could arise if nutrient/waste exchange across the plasma membrane is a limiting factor for growth. We observed that red light-illuminated optoBem1 cells also exhibited a change in DNA content over time. While most cells maintained a ploidy of 2N or less during the first 3 h of Bem1 disruption, a population of 4N cells appeared following 6 h of arrest , consistent with prior reports suggesting that after Bem1 disruption, some arrested cells eventually leak through the cell cycle block and undergo DNA endoreduplication. To ensure that the surface area-proportional growth was not an artifact of increased ploidy, we set out to generate ‘giant yeast’ via a second, non-optogenetic method: disruption of Cdk1/Cdc28 using the temperature-sensitive allele cdc28-13 . Unlike optoBem1 cells, nearly all cdk1-ts cells at the restrictive temperature arrest in G1 without undergoing further DNA replication. We found that cdk1-ts cells grown at the restrictive temperature to induce arrest in G1 also exhibited a linear increase in cell diameter, consistent with growth proportional to surface area . However, cdk1-ts were unable to maintain this rate of growth over the entire 12-h time-course: After reaching a volume of 500–700 μm3 , cell growth stalled . Taken together, our results from both optoBem1 and cdk1-ts cells indicate that the isotropic growth rate during G1 is proportional to surface area over a wide range of cell sizes.