Transcripts switched off at the stop of phloem unloading highlight the energy efficiency of sugar import in the ripening V. vinifera fruit

Transcriptomic changes at the cessation of sugar accumulation in the pericarp of Vitis vinifera were addressed on single berries re-synchronised according to their individual growth patterns. The net rates of water, sugars and K+ accumulation inferred from individual growth and solute concentration confirmed that these inflows stopped simultaneously in the ripe berry, while the small amount of malic acid remaining at this stage was still being oxidised at low rate. Re-synchronised individual berries displayed negligible variations in gene expression among triplicates. RNA-seq studies revealed sharp reprogramming of cell-wall enzymes and structural proteins at the stop of phloem unloading, associated with an 80% repression of multiple sugar transporters and aquaporins on the plasma or tonoplast membranes, with the noticeable exception of H+/sugar symporters, which were rather weakly and constitutively expressed. This was verified in three genotypes placed in contrasted thermo-hydric conditions. The prevalence of SWEET suggests that electrogenic transporters would play a minor role on the plasma membranes of SE/CC complex and the one of the flesh, while sucrose/H+ exchangers dominate on its tonoplast. Cis-regulatory elements present in their promoters allowed to sort these transporters in different groups, also including specific TIPs and PIPs paralogs, and cohorts of cell wall-related genes. Together with simple thermodynamic considerations, these results lead to propose that H+/sugar exchangers at the tonoplast, associated with a considerably acidic vacuolar pH, may exhaust cytosolic sugars in the flesh and alleviate the need for supplementary energisation of sugar transport at the plasma membrane.

Hexose translocation mediated by SlSWEET5b is required for pollen maturation in Solanum lycopersicum

Pollen fertility is critical for successful fertilization and, accordingly, for crop yield. While sugar unloading affects growth and development of all types of sink organs, the molecular nature for sugar import to tomato pollen is poorly understood. However, SWEET transporters have been proposed to function in pollen development. Here, qRT-PCR revealed that SlSWEET5b was markedly expressed in flowers when compared to the remaining tomato SlSWEETs; particularly, in the stamens of maturing flower buds undergoing mitosis. Distinct accumulation of SlSWEET5b-GUS fusion proteins was present in mature flower buds, especially in anther vascular and inner cells, symplasmic isolated pollen cells and styles. The demonstration that GFP fusion proteins located to the plasma membrane support the idea that the SlSWEET5b carrier functions in apoplasmic sugar translocation during pollen maturation. Such function is in line with data from yeast complementation experiments and radiotracer uptakes, showing that SlSWEET5b operates as a low affinity hexose-specific passive facilitator, with a KM of ~36 mM. Most importantly, RNAi-mediated suppression of SlSWEET5b expression resulted in shrunken nucleus-less pollen cells, impaired germination and low seed yield. Interestingly, stamens from SlSWEET5b-silenced tomato mutants contained significantly lower amounts of sucrose and increased invertase activity, pointing to reduced carbon supply and perturbed sucrose homeostasis in this tissue. Taken together, our findings reveal an essential role of SlSWEET5b in mediating apoplasmic hexose import into phloem unloading cells and into developing pollen cells to support pollen mitosis and maturation in tomato flowers.One-sentence SummaryPlasma-membrane-localized SlSWEET5b facilitates a sequential hexose flux, from phloem to anther cells and from anther locule to pollen, to support pollen maturation and fertility in tomato flowers.

Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem are required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Coupling kinetic models and advection-diffusion equations. 2. Sensitivity analysis of an advection-diffusion-reaction model

The accompanying paper (Uys et al., in silico Plants, XXXX) presented a core model of sucrose accumulation within the advection-diffusion-reaction framework, which is able to capture the spatio-temporal evolution of the system from a set of initial conditions. This paper presents a sensitivity analysis of this model. Because this is a non-steady-state model based on partial differential equations, we performed the sensitivity analysis using two approaches from engineering. The Morris method is based on a one-at-a-time design, perturbing parameters individually and calculating the influence on model output in terms of elementary effects. FAST is a global sensitivity analysis method, where all parameters are perturbed simultaneously, oscillating at different frequencies, enabling the calculation of the contribution of each parameter through Fourier analysis. Overall, both methods gave similar results. Perturbations in reactions tended to have a large influence on their own rate, as well as on directly connected metabolites. Sensitivities varied both with the time of the simulation and the position along the sugarcane stalk. Our results suggest that vacuolar sucrose concentrations are most sensitive to vacuolar invertase in the centre of the stalk, but that phloem unloading and vacuolar sucrose uptake also contribute, especially towards the stalk edges. Sucrose in the phloem was most sensitive to phloem loading at the nodes, but most sensitive to phloem unloading in the middle of the internodes. Sink concentrations of sucrose in the symplast were most sensitive to phloem unloading in the middle of the internodes, but at the nodes cytosolic invertase had the greatest effect.

Auxin-Mediated Regulation of Dorsal Vascular Cell Development May Be Responsible for Sucrose Phloem Unloading in Large Panicle Rice

Large panicle rice cultivars often fail to fulfill their high-yield potential due to the poor grain filling of inferior spikelets (IS), which appears as initially stagnant development and low final seed weight. Understanding the mechanism of the initial stagnancy is important to improve IS grain filling. In this study, superior spikelets (SS) were removed from two homozygous japonica rice varieties (W1844 and CJ03) with the same sink capacity in an attempt to force photosynthate transport to the IS. The results showed that SS removal increased the grain weight, sucrose content, starch accumulation, and endogenous IAA levels of IS during the initial grain-filling stage. SS removal also improved the patterns of vascular cells in the dorsal pericarp and the expression levels of genes involved in sucrose transport (OsSUTs and OsSWEETs) and IAA metabolism (OsYUCs and OsPINs). Exogenous IAA application advanced the initiation of grain filling by increasing the sucrose content and the gene expression levels of sucrose transporters. These results indicate that auxin may act like a signal substance and play a vital role in initial grain filling by regulating dorsal vascular cell development and sucrose phloem unloading into caryopsis.

Transcripts repressed at the stop of phloem unloading highlight the energy efficiency of sugar import in the ripening V. vinifera fruit.

Transcriptomic changes at the cessation of sugar accumulation in the pericarp of Vitis vinifera were addressed on single berries re-synchronized according to their individual growth patterns. The net rates of water, sugars and K+ accumulation inferred from individual growth and solute concentration confirmed that these inflows stopped simultaneously in the ripe berry, while the small amount of malic acid remaining at this stage was still being oxidized at a low rate. Resynchronized individual berries displayed negligible variations in gene expression among triplicates. RNA-Seq studies revealed sharp reprogramming of cell wall enzymes and structural proteins, associated with an 80% repression of specific sugar transporters and aquaporins on the plasma or tonoplast membranes, at the stop of phloem unloading in the three genotypes and two environments investigated. The prevalence of SWEET transporters suggests that electrogenic transporters would just play a minor role on the plasma membrane of SE/CC complex, and the one of the flesh, while sucrose/H+ exchangers dominate on its tonoplast. Cis-regulatory elements present in their promoters allowed to sort these transporters in different groups, also including specific TIPs and PIPs paralogs, and cohorts of cell wall related genes. These results lead us to propose which structural, developmental and energy adaptations would give this fruit such a power of attraction for water and photoassimilates.