Transcriptional regulation of bark freezing tolerance in apple (Malus domestica Borkh.)

Freezing tolerance is a significant trait in plants that grow in cold environments and survive through the winter. Apple (Malus domestica Borkh.) is a cold-tolerant fruit tree, and the cold tolerance of its bark is important for its survival at low temperatures. However, little is known about the gene activity related to its freezing tolerance. To better understand the gene expression and regulation properties of freezing tolerance in dormant apple trees, we analyzed the transcriptomic divergences in the bark from 1-year-old branches of two apple cultivars, “Golden Delicious” (G) and “Jinhong” (H), which have different levels of cold resistance, under chilling and freezing treatments. “H” can safely overwinter below −30 °C in extremely low-temperature regions, whereas “G” experiences severe freezing damage and death in similar environments. Based on 28 bark transcriptomes (from the epidermis, phloem, and cambium) from 1-year-old branches under seven temperature treatments (from 4 to −29 °C), we identified 4173 and 7734 differentially expressed genes (DEGs) in “G” and “H”, respectively, between the chilling and freezing treatments. A gene coexpression network was constructed according to this expression information using weighted gene correlation network analysis (WGCNA), and seven biologically meaningful coexpression modules were identified from the network. The expression profiles of the genes from these modules suggested the gene regulatory pathways that are responsible for the chilling and freezing stress responses of “G” and/or “H.” Module 7 was probably related to freezing acclimation and freezing damage in “H” at the lower temperatures. This module contained more interconnected hub transcription factors (TFs) and cold-responsive genes (CORs). Modules 6 and 7 contained C-repeat binding factor (CBF) TFs, and many CBF-dependent homologs were identified as hub genes. We also found that some hub TFs had higher intramodular connectivity (KME) and gene significance (GS) than CBFs. Specifically, most hub TFs in modules 6 and 7 were activated at the beginning of the early freezing stress phase and maintained upregulated expression during the whole freezing stress period in “G” and “H”. The upregulation of DEGs related to methionine and carbohydrate biosynthetic processes in “H” under more severe freezing stress supported the maintenance of homeostasis in the cellular membrane. This study improves our understanding of the transcriptional regulation patterns underlying freezing tolerance in the bark of apple branches.

Prevention of ice propagation by permeability barriers in bud axes of Vitis vinifera

A physical barrier exists in Vitis vinifera L. (wine-quality grapevines) between the canes that freeze at subzero temperatures and the buds that supercool, thereby avoiding ice formation in these delicate organs. Our objective was to characterize the ice nucleation barrier by observing grape buds at different stages of acclimation, by treating them with pectinase, and by comparing them to buds of the more hardy Vitis riparia L. and to non-supercooling buds of Populus nigra L. "Italica." Differential thermal analysis (DTA) indicated that as V. vinifera andV. riparia acclimated in the autumn, the ice nucleation point of the buds declined, then increased as the grapevines de-acclimated in the spring. Laser scanning confocal microscopy was used to detect the penetration of fluorescent rhodamine green dyes from the cane into the bud as a measure of apoplastic permeability. These pore size exclusion tests indicated that the bud axes were impermeable to a 3000 MW dextran-conjugated dye when the ice nucleation temperature was below -20°C, but permeable above -20°C. This lower porosity presumably restricts the penetration of ice into the bud from the cane. In contrast to the two Vitis species, the permeability of Populus buds did not limit ice propagation, and the water in buds froze between -5 and -10°C according to DTA. Pectinase and phosphate treatments of grape buds to remove pectin increased apoplastic permeability, increased the ice nucleation temperature, and prevented supercooling according to DTA. Light microscopy indicated that suberin coated the scales of the grape buds, probably preventing ice nucleation from the environment, but was not present in the bud axis region. We concluded that a permeability barrier, possibly containing pectin, in the axis of grape buds limits the propagation of ice into the buds and enables supercooling to occur.Key words: supercooling, freezing, acclimation, winter hardiness, pectin, confocal microscopy.

Irrigation Scheduling, Nutrient Application Frequency, and Freezing Acclimation of `Hamlin' Orange Trees in Florida

`Hamlin' orange trees [C. sinensis (L.) Osb.] from a commercial nursery were planted into raised beds on a site that simulated conditions typical of the flatwoods region of the citrus industry. A factorial experiment with three irrigation schedules, based on growth flushes and three nutrient application frequencies (total N, 0.136 kg/tree per year), was conducted in 1994. Trees were irrigated using 90° microsprinklers, and soil moisture content was monitored using a neutron probe. Eleven replicate trees of the nine treatments were included in a completely randomized block design. Weekly freeze tests using the electrolyte leakage method were conducted at –4, –6, and –8C. Electrolyte leakage was determined using a conductivity meter. Different irrigation scheduling based on growth flushes had no significant effect on freezing acclimation. However, increased frequency and lower amounts of fertilizer per application significantly (P = 0.05) increased freeze hardiness from 4.2 to –6.10C by the end of November. Morphological data including trunk diameter, tree height, and flushing status also were recorded. Increasing frequency of nutrient application resulted in a more rapid acclimation of young `Hamlin' orange trees.