An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Desiree' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4 degrees C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response

The Flowering Integrator FT Regulates SEPALLATA3 and FRUITFULL Accumulation in Arabidopsis Leaves

The transition to flowering involves major changes in the shoot apical meristem and in the fate of existing leaf primordia. Transcripts of the Arabidopsis thaliana flowering-promoting gene FLOWERING LOCUS T (FT) are present in leaf tissue but can also promote flowering when artificially introduced into the meristem. FT may normally act in the leaf and/or the meristem, initiating or constituting a mobile flower-promoting signal. We studied FT-dependent events in the rosette leaf, some of which might precede or mimic events in the meristem and its primordia. We show FT-dependent transcript accumulation of the MADS box transcription factors FRUITFULL (FUL) and SEPALLATA3 (SEP3) in leaves. Abnormally high levels of FT further increase the expression of these genes, leading to morphological changes in the leaves. Loss of the flowering-time gene FD, as well as environmental conditions that delay flowering, reduce FT's effect on leaves via reduced activation of its targets. FUL, SEP3, and APETALA1 accumulation in the meristem is associated with and contributes to the transition to flowering. We propose that FT functions through partner-dependent transcriptional activation of these and as-yet-unknown genes and that this occurs at several sites. Organ fate may depend on both degree of activation and the developmental stage reached by the organ before activation occurs

The Role of Aquaporins in pH-Dependent Germination of Rhizopus delemar Spores.

Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an up to 3-fold increase in spore diameter, whereas spore diameter was smaller in a neutral environment. When suspended in an acidic environment, the spores started to float, indicating a change in their density. Treatment of the spores with HgCl2, an aquaporin blocker, prevented floating and inhibited spore swelling and germ-tube emergence, indicating the importance of water uptake at the early stages of germination. Two putative candidate aquaporin-encoding genes-RdAQP1 and RdAQP2-were identified in the R. delemar genome. Both presented the conserved NPA motif and six-transmembrane domain topology. Expressing RdAQP1 and RdAQP2 in Arabidopsis protoplasts increased the cells' osmotic water permeability coefficient (Pf) compared to controls, indicating their role as water channels. A decrease in R. delemar aquaporin activity with increasing external pH suggested pH regulation of these proteins. Substitution of two histidine (His) residues, positioned on two loops facing the outer side of the cell, with alanine eliminated the pH sensing resulting in similar Pf values under acidic and basic conditions. Since hydration is critical for spore switching from the resting to activate state, we suggest that pH regulation of the aquaporins can regulate the initial phase of R. delemar spore germination, followed by germ-tube elongation and host-tissue infection

Stronger sink demand for metabolites supports dominance of the apical bud in etiolated growth

The potato tuber is a swollen underground stem that can sprout under dark conditions. Sprouting initiates in the tuber apical bud (AP), while lateral buds (LTs) are repressed by apical dominance (AD). Under conditions of lost AD, removal of tuber LTs showed that they partially inhibit AP growth only at the AD stage. Detached buds were inhibited by exogenous application of naphthaleneacetic acid (NAA), whereas 6-benzyladenine (6-BA) and gibberellic acid (GA3) induced bud burst and elongation, respectively. NAA, applied after 6-BA or GA3, nullified the latters' growth-stimulating effect in both the AP and LTs. GA3 applied to the fifth-position LT was transported mainly to the tuber's AP. GA3 treatment also resulted in increased indole-3-acetic acid (IAA) concentration and cis-zeatin O-glucoside in the AP. In a tuber tissue strip that included two or three buds connected by the peripheral vascular system, treatment of a LT with GA3 affected only the AP side of the strip, suggesting that the AP is the strongest sink for GA3, which induces its etiolated elongation. Dipping etiolated sprouts in labeled GA3 showed specific accumulation of the signal in the AP. Transcriptome analysis of GA3's effect showed that genes related to the cell cycle, cell proliferation, and hormone transport are up-regulated in the AP as compared to the LT. Sink demand for metabolites is suggested to support AD in etiolated stem growth by inducing differential gene expression in the AP

Effect of major peaks resulting from HPLC fractionation of sweet potato active fraction (SPAF) on <i>Rhizopus delemar</i> spore swelling and germination.

<p>The pH of each HPLC fraction was modified to 4.7 or 7. The peaks contained: 1 –a mixture of organic and amino acids, 2 –sucrose, 3- glucose, 4 –fructose, or the combination of 1 and 3. SPAF and water served as controls. Values are means ± SE (n = 500).</p

Effect of HgCl₂, an inhibitor of AQPs function on swelling of spore of <i>Rhizopus delemar</i>.

<p>Treatment of <i>R</i>. <i>delemar</i> spores with 40 μM HgCl₂ for 5 min inhibited spore swelling and germination. Additional treatment (5 min) with 5 μM of the reducing agent 2-β-mercaptoethanol (2ME) fully reversed the inhibition effect. Pictures were taken 15 min and 3 h, in the upper and lower rows, respectively, after incubation in water or sweet potato active fraction (SPAF). Bar = 100 μm.</p

Effect of HgCl₂ on swelling of <i>Rhizopus delemar</i> spores incubated in sweet potato active fraction (SPAF) and germ-tube emergence.

<p>Spores were treated with 40 μM HgCl₂ to inhibit aquaporin activity. Additional treatment with the reducing agent 2-β-mercaptoethanol (2ME, 5 μM) was used to reverse the HgCl₂ inhibition effect. The assays were performed at pH 4.7. Values are means ± SE (n = 500)</p

Phylogenetic tree of 231 fungal major intrinsic proteins (MIPs).

<p>The MIPs clustered into four distinct groups: Cluster I—putative water channels MIPs (represented by ADC55259|<i>Saccharomyces cerevisiae</i> [Black circle] and JF491353|<i>Terfezia claveryi</i> [black square]), cluster II—putative aquaglyceroporins MIPs that preferentially transporting small neutral molecules (represented by Lacbi2|671860|<i>Laccaria bicolor</i> [open triangle]), cluster III—MIPs that putatively act as water and small neutral molecule transport channels (represented by GAA23030|<i>S</i>. <i>cerevisiae</i> [black rhombus]), and cluster IV—putative fungal X intrinsic proteins (XIPs) (represented by TmeAQP2|<i>Tuber melanosporum</i> [black triangle]). Both <i>RdAQP1</i> and <i>RdAQP2</i> (open circles) are located in Cluster III, pointing on their potential capability to act as a water channels. Bar represents 0.2 changes.</p

Effect of pH on <i>Rhizopus delemar</i> spore germination.

<p>Percentage of <i>R</i>. <i>delemar</i> spores germinated under different pH conditions. Spores were incubated in SPAF solution (20 mg/ml, 42°C) and scored at 6 h. Values are means ± SE (n = 500).</p