Arabidopsis Sucrose Transporter AtSuc1 introns act as strong enhancers of expression

The expression of AtSUC1 is controlled by the promoter and intragenic sequences. AtSUC1 is expressed in roots, pollen and trichomes. However, AtSUC1 promoter-GUS transgenics only show expression in trichomes and pollen. Here, we show that the root expression of AtSUC1 is controlled by an interaction between the AtSUC1 promoter and two short introns. The deletion of either intron from whole-gene-GUS constructs results in no root expression, showing that both introns are required. The two introns in tandem, fused to GUS, produce high constitutive expression throughout the vegetative parts of the plant. When combined with the promoter, the expression driven by the introns is reduced and localized to the roots. In Arabidopsis seedlings, exogenously applied sucrose induces the expression of AtSUC1 in roots and causes anthocyanin accumulation. atsuc1 loss-of-function mutants are defective in sucrose-induced anthocyanin accumulation. We show that an AtSUC1 whole-gene-GUS construct expressing a nonfunctional AtSUC1 (D152N) mutant, that is transport inactive, is defective in sucrose-induced AtSUC1 expression when expressed in an atsuc1-null background. We also show that the transport-defective allele does not complement the loss of sucrose-induced anthocyanin accumulation in null atsuc1 mutants. The results indicate that sucrose uptake via AtSUC1 is required for sucrose-induced AtSUC1 expression and sucrose-induced anthocyanin accumulation and that the site for sucrose detection is intracellular.

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development

Overexpression of the HvCslF6 gene in hull-less barley grain perturbs sucrose uptake and allocation, and impairs transfer tissue and endosperm development

Immediate inhibition of sucrose uptake inCorynebacterium glutamicumin response to intracellular glucose-6-phosphate accumulation requires theptsGencoded EII-permease

ABSTRACTCorynebacterium glutamicumco-metabolizes most carbon sources, such as glucose and sucrose. Uptake of those sugars by the PTS involves a glucose- and a sucrose-specific permease EIIGlc(ptsG) and EIISuc(ptsS), respectively. Block of glycolysis by deletion ofpgi(encodes phosphoglucoisomerase) redirects glucose-driven carbon flux towards pentose phosphate pathway.C. glutamicumΔpgigrows poorly with glucose but has unaffected, good growth with sucrose. However, addition of glucose to sucrose-cultivatedC. glutamicumΔpgiimmediately arrested growth via inhibition of the EIISuc–mediated sucrose uptake and reduction ofptsS-mRNA amounts. Kinetic analyses revealed that sucrose uptake inhibition inC. glutamicumΔpgitook place within 15 s after glucose addition. We show that inhibition of PTS-mediated sucrose uptake occurs as direct response to glucose-6-P accumulation. Moreover, addition of non-PTS substrates, which are metabolized to glucose-6-P such as maltose or glucose-6-P itself (uptake was enabled by heterologously produced UhpT), led to similar growth and sucrose uptake inhibition as glucose addition. Despite EIIGlcnot being involved in uptake of these substrates, negative effects on sucrose uptake after addition of maltose and glucose-6-P were absent in the EIIGlc–deficient strainC. glutamicumΔpgiΔptsG. These results show that theptsG-encoded EIIGlcis part of a novel mechanism for perception of intracellular glucose-6-P accumulation and instantaneous inhibition of EIISuc-mediated sucrose uptake inC. glutamicum. This novel mode of control of PTS activity by an early glycolytic metabolite probably allows efficient adaptation of sugar uptake to the capacity of the central metabolism during co-metabolization, which is characteristic forC. glutamicum.IMPORTANCECoordination of substrate uptake and metabolism are a prerequisite for efficient co-utilization of substrates, a trait typical for the Gram-positiveC. glutamicum. Sucrose uptake via the PTS permease EIISucin this organism immediately was inhibited in response to intracellular accumulation of the glycolysis intermediate glucose-6-phosphate. This inhibition depends exclusively on the presence but not activity of the PTS permease EIIGluc. Thus,C. glutamicumpossesses a novel, immediate, and PTS-dependent way to control and coordinate both uptake and metabolization of multiple substrates by monitoring of their metabolic levels in the cell. This offers new insights and interesting concepts for a further rational engineering of this industrially important production organism and exemplifies a putative general strategy of bacteria for the coordination of sugar uptake and central metabolism.

Extended Utilization of Constraint-Based Metabolic Model in a Long-Growing Crop

The constraint-based rMeCBM-KU50 model of cassava storage root growth was analyzed to evaluate its sensitivity, with respect to reaction flux distribution and storage root growth rate, to changes in model inputted data and constraints, including sucrose uptake rate-related data—photosynthetic rate, total leaf area, total photosynthetic rate, storage root dry weight, and biomass function-related data. These mainly varied within ±90% of the model default values, although exceptions were made for the carbohydrate (−90% to 8%) and starch (−90% to 9%) contents. The results indicated that the predicted storage root growth rate was highly affected by specific sucrose uptake rates through the total photosynthetic rate and storage root dry weight variations; whereas the carbon flux distribution, direction and partitioning inclusive, was more sensitive to the variation in biomass content, particularly the carbohydrate content. This study showed that the specific sucrose uptake rate based on the total photosynthetic rate, storage root dry weight, and carbohydrate content were critical to the constraint-based metabolic modeling and deepened our understanding of the input–output relationship—specifically regarding the rMeCBM-KU50 model—providing a valuable platform for the modeling of plant metabolic systems, especially long-growing crops.

Concomitant Loss of the Glyoxalase System and Glycolysis Makes the Uncultured Pathogen “Candidatus Liberibacter asiaticus” an Energy Scavenger

ABSTRACT Methylglyoxal (MG) is a cytotoxic, nonenzymatic by-product of glycolysis that readily glycates proteins and DNA, resulting in carbonyl stress. Glyoxalase I and II (GloA and GloB) sequentially convert MG into d-lactic acid using glutathione (GSH) as a cofactor. The glyoxalase system is essential for the mitigation of MG-induced carbonyl stress, preventing subsequent cell death, and recycling GSH for maintenance of cellular redox poise. All pathogenic liberibacters identified to date are uncultured, including “Candidatus Liberibacter asiaticus,” a psyllid endosymbiont and causal agent of the severely damaging citrus disease “huanglongbing.” In silico analysis revealed the absence of gloA in “Ca. Liberibacter asiaticus” and all other pathogenic liberibacters. Both gloA and gloB are present in Liberibacter crescens, the only liberibacter that has been cultured. L. crescens GloA was functional in a heterologous host. Marker interruption of gloA in L. crescens appeared to be lethal. Key glycolytic enzymes were either missing or significantly downregulated in “Ca. Liberibacter asiaticus” compared to (cultured) L. crescens. Marker interruption of sut, a sucrose transporter gene in L. crescens, decreased its ability to take up exogenously supplied sucrose in culture. “Ca. Liberibacter asiaticus” lacks a homologous sugar transporter but has a functional ATP/ADP translocase, enabling it to thrive both in psyllids and in the sugar-rich citrus phloem by (i) avoiding sucrose uptake, (ii) avoiding MG generation via glycolysis, and (iii) directly importing ATP from the host cell. MG detoxification enzymes appear to be predictive of “Candidatus” status for many uncultured pathogenic and environmental bacteria. IMPORTANCE Discovered more than 100 years ago, the glyoxalase system is thought to be present across all domains of life and fundamental to cellular growth and viability. The glyoxalase system protects against carbonyl stress caused by methylglyoxal (MG), a highly reactive, mutagenic and cytotoxic compound that is nonenzymatically formed as a by-product of glycolysis. The uncultured alphaproteobacterium “Ca. Liberibacter asiaticus” is a well-adapted endosymbiont of the Asian citrus psyllid, which transmits the severely damaging citrus disease “huanglongbing.” “Ca. Liberibacter asiaticus” lacks a functional glyoxalase pathway. We report here that the bacterium is able to thrive both in psyllids and in the sugar-rich citrus phloem by (i) avoiding sucrose uptake, (ii) avoiding (significant) MG generation via glycolysis, and (iii) directly importing ATP from the host cell. We hypothesize that failure to culture “Ca. Liberibacter asiaticus” is at least partly due to its dependence on host cells for both ATP and MG detoxification.

Inhibition of phloem loading by sulfite affects sugar distribution in pea leaves

Under the influence of sulfite, changes in the rate of ¹⁴CO₂ fixation, ¹⁴C-sucrose uptake and sugar content of the apoplastic and intracellular spaces of pea leaves varied according to the time of treatment and the anion concentration used. The greatest decrease of the rate of ¹⁴CO₂ and ¹⁴C-sucrose uptake in the presence of sulfite at concentrations from 0.1 to 5.0 mM was detected immediately (i.e. 5 min.) after supplying sulfite to the medium. Two hours later, a significant inhibition of photosynthesis and sucrose uptake was observed only with 5.0 and 2.5 and 5.0 mM concentrations of sulfite, respectively. The effect of sulfite (0.1-5.0 mM) on the apoplastic and intracellular sugar content for the first 30 min of treatment was negligible. However, after longer periods under sulfite treatment at concentrations ≥ 1.0 mM, significant increases in the apoplastic sucrose content were observed. An increase in the accumulation of starch was also noted. In light of our observations it suggested that the inhibition of phloem loading by sulfite induces disturbances in sugar distribution.

The growth and saponin production of Platycodon grandiflorum (Jacq.) A. DC. (Chinese bellflower) hairy roots cultures maintained in shake flasks and mist bioreactor

The growth and saponin accumulation were measured in two lines of transgenic hairy roots of <em>Platycodon grandiflorum</em>, Pl 6 and Pl 17, cultured for 8 weeks in 250-ml shake flasks containing 50 ml of hormone-free woody plant medium supplemented with 40 g/l sucrose and in the Pl 17 line cultured for 12 weeks in a 5-l mist bioreactor containing 1.5 l of the same medium. With both methods, the growth of transgenic hairy roots was assessed as both fresh and dry weight and the biomass growth was correlated with the conductivity and sucrose uptake. The accumulation of saponins was measured and compared with that in roots derived from the field cultivation. The saponin concentrations were significantly higher in the two hairy root lines cultured in shake flasks [6.92 g/100 g d.w. (g%) and 5.82 g% in Pl 6 and Pl 17, respectively] and the line cultured in the bioreactor (5.93 g%) than in the roots derived from the field cultivation (4.02 g%). The results suggest that cultures of <em>P. grandiflorum</em> hairy roots may be a valuable source for obtaining saponins.

Diverse response of tomato fruit explants to high temperature

Tomato explants (fruit with a pedicel and a piece of peduncle), with fruit growth stimulated by treating the flowers with NOA + GA&lt;sub&gt;3&lt;/sub&gt; (NG-series) were used as a model system for studying the effect of high temperature on C-sucrose uptake, its distribution and Ca retranslocation. Two cultivars with contrasting responses to high temperature were compared. In sensitive cv. Roma heat stress during 22h (40&lt;sup&gt;o&lt;/sup&gt;C for 10h and 30&lt;sup&gt;o&lt;/sup&gt;C for 12h), drastically depressed the uptake of ¹⁴C-sucrose coinciding with diminished fruit ¹⁴C-supply. It also decreased the specific activity of soluble acid invertase and the calcium content. All these strong negative responses to high temperature were markedly reduced in the NG-treated series involving remobilization of Ca to the fruits and a higher stability of the invertase activity. This indicates the indirect role of flower treatment with NG in addaptation to heat stress. In tolerant cv. Robin even higher temperatures (42&lt;sup&gt;o&lt;/sup&gt;C for 10h and 34&lt;sup&gt;o&lt;/sup&gt;C for 12h) were not stressful. They did not affect the ¹⁴C-sucrose uptake and stimulated ¹⁴C-supply to the fruit. Increased specific activity of acid invertase and a higher calcium content were also recorded but only in the control explants. In contrast to cv. Roma elevated temperature was slightly stressful for cv. Robin explants of NG-series. The differences in response of both cultivar explants to elevated temperature, based on unequal fruit supply with ¹⁴C-sucrose, seem to be causaly connected with two factors: the invertase activity being more or less sensitive to the heat stress, the ability to translocate Ca to the heated fruits.