Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.

MAIN CONCLUSION: An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

Vacuolar processing enzyme translocates to the vacuole through the autophagy pathway to induce programmed cell death.

The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.Abbreviations: ATG: autophagy related; CLP: caspase-like protease; HR: hypersensitive response; PCD: programmed cell death; St: Solanum tuberosum; VPE: vacuolar processing enzyme.

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance.

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem.

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.

Mint essential oil can induce or inhibit potato sprouting by differential alteration of apical meristem.

Sprouting of potatoes during storage, due to tuber dormancy release, is associated with weight loss and softening. Sprout-preventing chemicals, such as chlorpropham (CIPC), can negatively impact the environment and human health. Monthly thermal fogging with mint (Mentha spicata L.) essential oil (MEO) inhibited sprouting in eight potato cultivars during large-volume 6-month storage: the tubers remained firm with 38% lower weight loss after 140 days of storage. The sprout-inhibitory action may be nullified: treated tubers washed with water resumed sprouting within days, with reduced apical dominance. MEO application caused local necrosis of the bud meristem, and a few weeks later, axillary bud (AX) growth was induced in the same sprouting eye. MEO components analysis showed that 73% of its content is the monoterpene R-carvone. Tubers treated with synthetic R-carvone in equivalent dose, 4.5 microl l(-1), showed an inhibitory effect similar to that of MEO. Surprisingly, 0.5 microl l(-1) of MEO or synthetic R-carvone catalyzed AX sprouting in the tuber. To the best of our knowledge, this is the first report of an essential oil vapor inducing early sprouting of potato tubers. R-carvone caused visible damage to the meristem membrane at sprout-inhibiting, but not sprout-inducing doses, suggesting different underlying mechanisms. After 5 days' exposure to R-carvone, its derivatives transcarveol and neo-dihydrocarveol were found in buds of tubers treated with the inhibitory dose, suggesting biodegradation. These experiments demonstrate the potential of MEO vapor as an environmentally friendly alternative to CIPC in stored potatoes and as a research tool for the control of sprouting in plants.