Resistant starch is a popular nutritional additive to produce food with enhanced quality attributes, i.e., higher fiber content, and starchy horticultural commodities are similarly attractive. Te yield penalty of high amylose crops may be alleviated by picking an ideal AP/AM ratio through a coordinate change in the relative balance of starch biosynthetic enzymes. In the case of potato, it is plausible that down regulation of SBEs not only produces healthy fiber-starch, but also lessens the CIS severity and acrylamide problem . However, the sugars derived from starch during CIS may be an adaptive mechanism to enhance plant chilling tolerance. Rapid sugar accumulation upon cold stress have been reported in fruit. Te sugars freed from starch may promote metabolic activity and serve as an osmoprotectant, thus alleviating chilling injury. Te major functional SBEs were found to be upregulated in cold-stressed banana fruit, potato tuber, and Arabidopsis leaf , which may facilitate the ‘sugaring’ process. Modulating SBE activities may alter the rate of sugar released from the highly digestible starch polymers, thus changing the fruit/tuber cold responses. In fruiting species, the importance of ‘transitory-storage starch’ may be underestimated due to the lack of enough direct knowledge of its function, gained from experimental data. Tomato serves as a functional genomics model for feshy fruit, as it is easily transformed and genetically manipulated. Te putative function of ‘transitory-storage starch’ in fruit ripening, respiration, and sweetness enhancement may be revealed by engineering AP/AM ratio through over expression or suppression of SBEs. We hypothesize that high amylose, resistant starch tomato fruit may have reduced available starch, sugars,nft hydroponic system and changes in fruit ripening and other processes that are dependent on starch as a carbon supply and source of energy post harvest.
Tomato SBEs may not reflect the functionality of all fruit SBEs, but it would produce fundamental knowledge and expand our understanding of species-, organ- and developmental-specific regulations of the core starch biosynthetic enzymes.Starch, in general, plays an essential role in balancing the plant’s carbon budget as a reserve of glucose that is tightly related to sucrose metabolism and sugar signaling pathways.Starch is considered as an integrative mediator throughout the plant life cycle, regulating plant vegetative growth, reproductive growth, maturation and senescence, and response to abiotic stresses. This comprehensive regulation is achieved by changes in the synthesis and degradation of starch to balance glucose levels, after developmental and environmental triggers in different organs. Transitory starch and its biosynthesis have been well studied in the model plant Arabidopsis, but little research has been conducted on post harvest leafy greens. Quality metrics such as shelf-life, favor, color, firmness, and texture are of consumers’ choice, and they are related to the limited pools of storage compounds in detached leaves, which cells rely on to maintain basic cellular activities. A hypothesized function for the starch in packaged leaves could be presented as such: starch may act as a buffer against sugar starvation, and protect against cellular autophagy, by serving as an alternative energy source . If the biosynthesis and degradation of starch could be adjusted in a controlled way, then the modulated release of sugars may influence the post harvest shelf-life in detached leafy greens . A continuous, paced supply of sugars may preserve vacuolar nutrients and water content, leaf cellular structure and integrity, and, thus extend the ‘best by’ post harvest date of the produce. Although the eco-physiological role of amylose is poorly understood in Arabidopsis, the AP/AM ratio may set a threshold for the optimum usage of starch.
SBE action in leafy crops may differ from those in Arabidopsis given the dissimilar numbers of their isoforms and domain features . Modifying the quantity and quality of the starch in leafy greens such as spinach, lettuce, and watercress, by targeting starch biosynthetic enzymes, may provide evidence to its post harvest function in terms of produce longevity. Resistant starch is a popular nutritional additive to produce food with enhanced quality attributes, i.e., higher fiber content, and starchy horticultural commodities are similarly attractive. Te yield penalty of high amylose crops may be alleviated by picking an ideal AP/AM ratio through a coordinate change in the relative balance of starch biosynthetic enzymes. In the case of potato, it is plausible that down regulation of SBEs not only produces healthy fiber-starch, but also lessens the CIS severity and acrylamide problem . However, the sugars derived from starch during CIS may be an adaptive mechanism to enhance plant chilling tolerance. Rapid sugar accumulation upon cold stress have been reported in fruit. Te sugars freed from starch may promote metabolic activity and serve as an osmoprotectant, thus alleviating chilling injury. Te major functional SBEs were found to be upregulated in cold-stressed banana fruit, potato tuber, and Arabidopsis leaf , which may facilitate the ‘sugaring’ process. Modulating SBE activities may alter the rate of sugar released from the highly digestible starch polymers, thus changing the fruit/tuber cold responses. In fruiting species, the importance of ‘transitory-storage starch’ may be underestimated due to the lack of enough direct knowledge of its function, gained from experimental data. Tomato serves as a functional genomics model for feshy fruit, as it is easily transformed and genetically manipulated. Te putative function of ‘transitory-storage starch’ in fruit ripening, respiration, and sweetness enhancement may be revealed by engineering AP/AM ratio through overexpression or suppression of SBEs. We hypothesize that high amylose, resistant starch tomato fruit may have reduced available starch, sugars, and changes in fruit ripening and other processes that are dependent on starch as a carbon supply and source of energy post harvest.
Tomato SBEs may not reflect the functionality of all fruit SBEs, but it would produce fundamental knowledge and expand our understanding of species-, organ- and developmental-specific regulations of the core starch biosynthetic enzymes.Cell size correlates strongly with key aspects of cell physiology, including organelle abundance and DNA ploidy. Maintenance of uniform size may also underlie the efficient functioning of tissues and organs. 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,mobile grow rack 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, 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.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 .