Abiotic Stress Triggers the Expression of Genes Involved in Protein Storage Vacuole and Exocyst-Mediated Routes

Adverse conditions caused by abiotic stress modulate plant development and growth by altering morphological and cellular mechanisms. Plants’ responses/adaptations to stress often involve changes in the distribution and sorting of specific proteins and molecules. Still, little attention has been given to the molecular mechanisms controlling these rearrangements. We tested the hypothesis that plants respond to stress by remodelling their endomembranes and adapting their trafficking pathways. We focused on the molecular machinery behind organelle biogenesis and protein trafficking under abiotic stress conditions, evaluating their effects at the subcellular level, by looking at ultrastructural changes and measuring the expression levels of genes involved in well-known intracellular routes. The results point to a differential response of the endomembrane system, showing that the genes involved in the pathway to the Protein Storage Vacuole and the exocyst-mediated routes are upregulated. In contrast, the ones involved in the route to the Lytic Vacuole are downregulated. These changes are accompanied by morphological alterations of endomembrane compartments. The data obtained demonstrate that plants’ response to abiotic stress involves the differential expression of genes related to protein trafficking machinery, which can be connected to the activation/deactivation of specific intracellular sorting pathways and lead to alterations in the cell ultrastructure.

The Trafficking Machinery of Lytic and Protein Storage Vacuoles: How Much is Shared and How Much is Distinct?

Plant cells contain two types of vacuoles, the lytic vacuole and the protein storage vacuole. Lytic vacuoles (LVs) are present in vegetative cells, whereas protein storage vacuoles (PSVs) are found in seed cells. The physiological functions of the two vacuole types differ. Newly synthesized proteins must be transported to these vacuoles via protein trafficking through the endomembrane system for them to function. Recently, significant advances have been made in elucidating the molecular mechanisms of protein trafficking to these organelles. Despite these advances, the relationship between the trafficking mechanisms in LV and PSVs remains unclear. Some aspects of the trafficking mechanisms are common to both organelles, but certain aspects are specific to trafficking to either LV or PSVs. In this review, we summarize recent findings on the components involved in protein trafficking to both LV and PSVs and compare them to examine the extent of overlap in the trafficking mechanisms. In addition, we discuss the interconnection between the LV and PSVs in protein trafficking machinery and the implication in the identity of these organelles.

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic Nicotiana tabacum by Protein Storage Vacuole Targeting

Plant expression of microbial Cell Wall Degrading Enzymes (CWDEs) is a valuable strategy to produce industrial enzymes at affordable cost. Unfortunately, the constitutive expression of CWDEs may affect plant fitness to variable extents, including developmental alterations, sterility and even lethality. In order to explore novel strategies for expressing CWDEs in crops, the cellobiohydrolase CBM3GH5, from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus, was constitutively expressed in N. tabacum by targeting the enzyme both to the apoplast and to the protein storage vacuole. The apoplast targeting failed to isolate plants expressing the recombinant enzyme despite a large number of transformants being screened. On the opposite side, the targeting of the cellobiohydrolase to the protein storage vacuole led to several transgenic lines expressing CBM3GH5, with an enzyme yield of up to 0.08 mg g DW−1 (1.67 Units g DW−1) in the mature leaf tissue. The analysis of CBM3GH5 activity revealed that the enzyme accumulated in different plant organs in a developmental-dependent manner, with the highest abundance in mature leaves and roots, followed by seeds, stems and leaf ribs. Notably, both leaves and stems from transgenic plants were characterized by an improved temperature-dependent saccharification profile.

The PIF1-MIR408-Plantacyanin Repression Cascade Regulates Light Dependent Seed Germination

ABSTRACTLight-sensing seed germination is a vital process for the seed plants. A decisive event in light-induced germination is degradation of the central repressor PHYTOCHROME INTERACTING FACTOR1 (PIF1). It is also known that the balance between gibberellic acid (GA) and abscisic acid (ABA) critically controls germination. But the cellular mechanisms linking PIF1 turnover to hormonal rebalancing remain elusive. Here, employing far-red light-induced Arabidopsis seed germination as the experimental system, we identified Plantacyanin (PLC) as an inhibitor of germination, which is a storage vacuole-associated blue copper protein highly expressed in mature seed and rapidly silenced during germination. Molecular analyses showed that PIF1 directly binds to the MIR408 promoter and represses miR408 accumulation, which in turn post-transcriptionally modulates PLC abundance, thus forming the PIF1-MIR408-PLC repression cascade for translating PIF1 turnover to PLC turnover during early germination. Genetic analysis, RNA-sequencing, and hormone quantification revealed that PLC is necessary and sufficient to maintain the PIF1-mediated seed transcriptome and the low-GA-high-ABA state. Furthermore, we found that PLC domain organization and regulation by miR408 are conserved features in seed plants. These results unraveled a cellular mechanism whereby PIF1-relayed external light signals are converted through PLC-based copper mobilization into internal hormonal profiles for controlling seed germination.

Arabidopsis ECHIDNA protein is involved in seed coloration, protein trafficking to vacuoles, and vacuolar biogenesis

Flavonoids are a major group of plant-specific metabolites that determine flower and seed coloration. In plant cells, flavonoids are synthesized at the cytosolic surface of the endoplasmic reticulum and are sequestered in the vacuole. It is possible that membrane trafficking, including vesicle trafficking and organelle dynamics, contributes to flavonoid transport and accumulation. However, the underlying mechanism has yet to be fully elucidated. Here we show that the Arabidopsis ECHIDNA protein plays a role in flavonoid accumulation in the vacuole and protein trafficking to the vacuole. We found defective pigmentation patterns in echidna seed, possibly caused by reduced levels of proanthocyanidins, which determine seed coloration. The echidna mutant has defects in protein sorting to the protein storage vacuole as well as vacuole morphology. These findings indicate that ECHIDNA is involved in the vacuolar trafficking pathway as well as the previously described secretory pathway. In addition, we found a genetic interaction between echidna and green fluorescent seed 9 (gfs9), a membrane trafficking factor involved in flavonoid accumulation. Our findings suggest that vacuolar trafficking and/or vacuolar development, both of which are collectively regulated by ECHIDNA and GFS9, are required for flavonoid accumulation, resulting in seed coat pigmentation.

Trafficking to the seed protein storage vacuole

The processing and subcellular trafficking of seed storage proteins is a critical area of physiological, agricultural and biotechnological research. Trafficking to the lytic vacuole has been extensively discussed in recent years, without substantial distinction from trafficking to the protein storage vacuole (PSV). However, despite some overlap between these pathways, there are several examples of unique processing and machinery in the PSV pathway. Moreover, substantial new data has recently come to light regarding the important players in this pathway, in particular, the intracellular NHX proteins and their role in regulating lumenal pH. In some cases, these new data are limited to genetic evidence, with little mechanistic understanding. As such, the implications of these data in the current paradigm of PSV trafficking is perhaps yet unclear. Although it has generally been assumed that the major classes of storage proteins are trafficked via the same pathway, there is mounting evidence that the 12S globulins and 2S albumins may be trafficked independently. Advances in identification of vacuolar targeting signals, as well as an improved mechanistic understanding of various vacuolar sorting receptors, may reveal the differences in these trafficking pathways.