Identification of ultrastructural and biochemical cuticular markers influencing temperature of ice nucleation in selected genotypes of corn.

Corn is an economically important yet frost-sensitive crop, injured at the moment of ice nucleation. However, the influence of autumn temperatures on subsequent ice nucleation temperature is unknown. A 10-day chilling treatment under phytotron conditions ("mild", 18/6°C) or ("extreme", 10/5°C) generated no-visible damage but induced changes in the cuticle of the four genotypes in this study. The putatively more cold hardy Genotypes 884 and 959 leaves nucleated at colder temperatures compared to the more sensitive Genotypes 675 and 275. After chilling treatment, all four genotypes displayed warmer ice nucleation temperatures, with Genotype 884 expressing the largest shift to warmer nucleation temperatures. Cuticular hydrophobicity reduced while cuticular thickness remained unchanged under the chilling treatment. By contrast, under five-week field conditions, cuticle thickness increased in all genotypes, with Genotype 256 expressing a significantly thinner cuticle. FTIR spectroscopy revealed increases in the spectral regions of cuticular lipids in all genotypes after phytotron chilling treatment, while those spectral regions decreased under field conditions. A total of 142 molecular compounds were detected, with 28 compounds significantly induced under either phytotron or field conditions. Of these, seven compounds were induced under both conditions (Alkanes C31-C33, Ester C44, C46, ß-amyrin, and triterpene). While clear differential responses were observed, chilling conditions preceding a frost modified physical and biochemical properties of the leaf cuticle under both phytotron and field conditions indicating this response is dynamic and could be a factor in selecting corn genotypes better adapted to avoiding frost with lower ice nucleation temperature.

Cold and exogenous calcium alter Allium fistulosum cell wall pectin to depress intracellular freezing temperatures.

De-methyl esterification of homogalacturonan and subsequent cross-linking with Ca2+ is hypothesized to enhance the freezing survival of cold acclimated plants by reducing the porosity of primary cell walls. To test this theory, we collected leaf epidermal peels from non- (23/18 °C) and cold acclimated (2 weeks at 12/4 °C) Japanese bunching onion (Allium fistulosum L.). Cold acclimation enhanced the temperature at which half the cells survived freezing injury by 8 °C (LT50 =-20 °C), and reduced tissue permeability by 70-fold compared with non-acclimated epidermal cells. These effects were associated with greater activity of pectin methylesterase (PME) and a reduction in the methyl esterification of homogalacturonan. Non-acclimated plants treated with 50 mM CaCl2 accumulated higher concentrations of galacturonic acid, Ca2+ in the cell wall, and a lower number of visible cell wall pores compared with that observed in cold acclimated plants. Using cryo-microscopy, we observed that 50 mM CaCl2 treatment did not lower the LT50 of non-acclimated cells, but reduced the lethal intracellular ice nucleation to temperatures observed in cold acclimated epidermal cells. We postulate that the PME-homogalacturonan-mediated reduction in cell wall porosity is integral to intracellular freezing avoidance strategies in cold acclimated herbaceous cells.

Arabidopsis Ubiquitin-Conjugating Enzymes UBC4, UBC5, and UBC6 Have Major Functions in Sugar Metabolism and Leaf Senescence.

The ubiquitin-conjugating enzyme (E2) is required for protein ubiquitination. Arabidopsis has 37 E2s grouped into 14 subfamilies and the functions for many of them are unknown. We utilized genetic and biochemical methods to study the roles of Arabidopsis UBC4, UBC5, and UBC6 of the E2 subfamily IV. The Arabidopsis ubc4/5/6 triple mutant plants had higher levels of glucose, sucrose, and starch than the control plants, as well as a higher protein level of a key gluconeogenic enzyme, cytosolic fructose 1,6-bisphosphatase 1 (cyFBP). In an in vitro assay, the proteasome inhibitor MG132 inhibited the degradation of recombinant cyFBP whereas ATP promoted cyFBP degradation. In the quadruple mutant ubc4/5/6 cyfbp, the sugar levels returned to normal, suggesting that the increased sugar levels in the ubc4/5/6 mutant were due to an increased cyFBPase level. In addition, the ubc4/5/6 mutant plants showed early leaf senescence at late stages of plant development as well as accelerated leaf senescence using detached leaves. Further, the leaf senescence phenotype remained in the quadruple ubc4/5/6 cyfbp mutant. Our results suggest that UBC4/5/6 have two lines of important functions, in sugar metabolism through regulating the cyFBP protein level and in leaf senescence likely through a cyFBP-independent mechanism.

Transcriptomic analysis of differentially expressed genes in leaves and roots of two alfalfa (Medicago sativa L.) cultivars with different salt tolerance.

BACKGROUND: Alfalfa (Medicago sativa L.) production decreases under salt stress. Identification of genes associated with salt tolerance in alfalfa is essential for the development of molecular markers used for breeding and genetic improvement. RESULT: An RNA-Seq technique was applied to identify the differentially expressed genes (DEGs) associated with salt stress in two alfalfa cultivars: salt tolerant 'Halo' and salt intolerant 'Vernal'. Leaf and root tissues were sampled for RNA extraction at 0 h, 3 h, and 27 h under 12 dS m- 1 salt stress maintained by NaCl. The sequencing generated a total of 381 million clean sequence reads and 84.8% were mapped on to the alfalfa reference genome. A total of 237 DEGs were identified in leaves and 295 DEGs in roots of the two alfalfa cultivars. In leaf tissue, the two cultivars had a similar number of DEGs at 3 h and 27 h of salt stress, with 31 and 49 DEGs for 'Halo', 34 and 50 for 'Vernal', respectively. In root tissue, 'Halo' maintained 55 and 56 DEGs at 3 h and 27 h, respectively, while the number of DEGs decreased from 42 to 10 for 'Vernal'. This differential expression pattern highlights different genetic responses of the two cultivars to salt stress at different time points. Interestingly, 28 (leaf) and 31 (root) salt responsive candidate genes were highly expressed in 'Halo' compared to 'Vernal' under salt stress, of which 13 candidate genes were common for leaf and root tissues. About 60% of DEGs were assigned to known gene ontology (GO) categories. The genes were involved in transmembrane protein function, photosynthesis, carbohydrate metabolism, defense against oxidative damage, cell wall modification and protection against lipid peroxidation. Ion binding was found to be a key molecular activity for salt tolerance in alfalfa under salt stress. CONCLUSION: The identified DEGs are significant for understanding the genetic basis of salt tolerance in alfalfa. The generated genomic information is useful for molecular marker development for alfalfa genetic improvement for salt tolerance.

Elucidation of molecular and hormonal background of early growth cessation and endodormancy induction in two contrasting Populus hybrid cultivars.

BACKGROUND: Over the life cycle of perennial trees, the dormant state enables the avoidance of abiotic stress conditions. The growth cycle can be partitioned into induction, maintenance and release and is controlled by complex interactions between many endogenous and environmental factors. While phytohormones have long been linked with dormancy, there is increasing evidence of regulation by DAM and CBF genes. To reveal whether the expression kinetics of CBFs and their target PtDAM1 is related to growth cessation and endodormancy induction in Populus, two hybrid poplar cultivars were studied which had known differential responses to dormancy inducing conditions. RESULTS: Growth cessation, dormancy status and expression of six PtCBFs and PtDAM1 were analyzed. The 'Okanese' hybrid cultivar ceased growth rapidly, was able to reach endodormancy, and exhibited a significant increase of several PtCBF transcripts in the buds on the 10th day. The 'Walker' cultivar had delayed growth cessation, was unable to enter endodormancy, and showed much lower CBF expression in buds. Expression of PtDAM1 peaked on the 10th day only in the buds of 'Okanese'. In addition, PtDAM1 was not expressed in the leaves of either cultivar while leaf CBFs expression pattern was several fold higher in 'Walker', peaking at day 1. Leaf phytohormones in both cultivars followed similar profiles during growth cessation but differentiated based on cytokinins which were largely reduced, while the Ox-IAA and iP7G increased in 'Okanese' compared to 'Walker'. Surprisingly, ABA concentration was reduced in leaves of both cultivars. However, the metabolic deactivation product of ABA, phaseic acid, exhibited an early peak on the first day in 'Okanese'. CONCLUSIONS: Our results indicate that PtCBFs and PtDAM1 have differential kinetics and spatial localization which may be related to early growth cessation and endodormancy induction under the regime of low night temperature and short photoperiod in poplar. Unlike buds, PtCBFs and PtDAM1 expression levels in leaves were not associated with early growth cessation and dormancy induction under these conditions. Our study provides new evidence that the degradation of auxin and cytokinins in leaves may be an important regulatory point in a CBF-DAM induced endodormancy. Further investigation of other PtDAMs in bud tissue and a study of both growth-inhibiting and the degradation of growth-promoting phytohormones is warranted.

Dissecting the Roles of Cuticular Wax in Plant Resistance to Shoot Dehydration and Low-Temperature Stress in Arabidopsis.

Cuticular waxes are a mixture of hydrophobic very-long-chain fatty acids and their derivatives accumulated in the plant cuticle. Most studies define the role of cuticular wax largely based on reducing nonstomatal water loss. The present study investigated the role of cuticular wax in reducing both low-temperature and dehydration stress in plants using Arabidopsis thaliana mutants and transgenic genotypes altered in the formation of cuticular wax. cer3-6, a known Arabidopsis wax-deficient mutant (with distinct reduction in aldehydes, n-alkanes, secondary n-alcohols, and ketones compared to wild type (WT)), was most sensitive to water loss, while dewax, a known wax overproducer (greater alkanes and ketones compared to WT), was more resistant to dehydration compared to WT. Furthermore, cold-acclimated cer3-6 froze at warmer temperatures, while cold-acclimated dewax displayed freezing exotherms at colder temperatures compared to WT. Gas Chromatography-Mass Spectroscopy (GC-MS) analysis identified a characteristic decrease in the accumulation of certain waxes (e.g., alkanes, alcohols) in Arabidopsis cuticles under cold acclimation, which was additionally reduced in cer3-6. Conversely, the dewax mutant showed a greater ability to accumulate waxes under cold acclimation. Fourier Transform Infrared Spectroscopy (FTIR) also supported observations in cuticular wax deposition under cold acclimation. Our data indicate cuticular alkane waxes along with alcohols and fatty acids can facilitate avoidance of both ice formation and leaf water loss under dehydration stress and are promising genetic targets of interest.

A single seed treatment mediated through reactive oxygen species increases germination, growth performance, and abiotic stress tolerance in Arabidopsis and rice.

Hydroxyl radical (•OH) is considered to be the most damaging among reactive oxygen species. Although afew studies have reported on its effects on growth and stress adaptation of plants, no detailed studies have been performed using •OH in germination and early seedling growth under abiotic stresses. Here we report a single seed treatment with •OH on germination and seedling growth of Arabidopsis and rice under non-stressed (ambient) and various abiotic-stressed conditions (chilling, high temperature, heat, and salinity). The treatment resulted in faster seed germination and early seedling growth under non-stressed conditions, and, interestingly, these effects were more prominent under abiotic stresses. In addition, Arabidopsis seedlings from treated seeds showed faster root growth and developed more lateral roots. These results show apositive and potential practical use for •OH in model and crop plants for direct seeding in the field, as well as improvement of tolerance against emerging stresses. Abbreviations: AUC: area under curve; MGT: mean germination time; t50: time to reach 50% germination; U7525: time for uniform germination from 25% to 75%; ROS: reactive oxygen species; GSI: germination speed index; SI: stress index; DI: dormancy index.

Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing.

Meaningful improvements in winter cereal cold hardiness requires a complete model of freezing behaviour in the critical crown organ. Magnetic resonance microimaging diffusion-weighted experiments provided evidence that cold acclimation decreased water content and mobility in the vascular transition zone (VTZ) and the intermediate zone in rye (Secale cereale L. Hazlet) compared with wheat (Triticum aestivum L. Norstar). Differential thermal analysis, ice nucleation, and localization studies identified three distinct exothermic events. A high-temperature exotherm (-3°C to -5°C) corresponded with ice formation and high ice-nucleating activity in the leaf sheath encapsulating the crown. A midtemperature exotherm (-6°C and -8°C) corresponded with cavity ice formation in the VTZ but an absence of ice in the shoot apical meristem (SAM). A low-temperature exotherm corresponded with SAM injury and the killing temperature in wheat (-21°C) and rye (-27°C). The SAM had lower ice-nucleating activity and freezing survival compared with the VTZ when frozen in vitro. The intermediate zone was hypothesized to act as a barrier to ice growth into the SAM. Higher cold hardiness of rye compared with wheat was associated with higher VTZ and intermediate zone desiccation resulting in the formation of ice barriers surrounding the SAM.

Tissue-specific changes in apoplastic proteins and cell wall structure during cold acclimation of winter wheat crowns.

The wheat (Triticum aestivum L.) crown is the critical organ of low temperature stress survival over winter. In cold-acclimated crowns, ice formation in the apoplast causes severe tissue disruption as it grows at the expense of intracellular water. While previous crown studies have shown the vascular transition zone (VTZ) to have a higher freezing sensitivity than the shoot apical meristem (SAM), the mechanism behind the differential freezing response is not fully understood. Cooling cold-acclimated crowns to -10 °C resulted in an absence of VTZ tetrazolium chloride staining, whereas the temperatures at which 50% of the SAM stained positive and 50% of plants recovered (LT50) were similar after cold acclimation for 21 (-16 °C) and 42 d (-20 °C) at 4 °C. Proteomic analysis of the apoplastic fluids identified dehydrins, vernalization-responsive proteins, and cold shock proteins preferentially accumulated in the SAM. In contrast, modifications to the VTZ centered on increases in pathogenesis-related proteins, anti-freeze proteins, and sugar hydrolyzing enzymes. Fourier transform infrared spectroscopy focal plane array analysis identified the biochemical modification of the cell wall to enhance methyl-esterified cross-linking of glucuronoarabinoxylans in the VTZ. These findings indicate that the SAM and VTZ express two distinct tissue-specific apoplastic responses during cold acclimation.

Wheat flag leaf epicuticular wax morphology and composition in response to moderate drought stress are revealed by SEM, FTIR-ATR and synchrotron X-ray spectroscopy.

Wheat (Triticum aestivum L.) is the largest cereal crop grown in Western Canada where drought during late vegetative and seed filling stages affects plant development and yield. To identify new physiochemical markers associated with drought tolerance, epidermal characteristics of the flag leaf of two wheat cultivars with contrasting drought tolerance were investigated. The drought resistant 'Stettler' had a lower drought susceptibility index, greater harvest index and water-use efficiency than the susceptible 'Superb'. Furthermore, flag leaf width, relative water content and leaf roll were significantly greater in Stettler than in Superb at moderate drought stress (MdS). Visible differences in epicuticular wax density on the adaxial flag leaf surfaces and larger bulliform cells were identified in Stettler as opposed to Superb. Mid-infrared attenuated total internal reflectance spectra revealed that Stettler flag leaves had increased asymmetric and symmetric CH2 but reduced carbonyl esters on its adaxial leaf surface compared to Superb under MdS. X-ray fluorescence spectra revealed a significant increase in total flag leaf Zn concentrations in Stettler in response to MdS. Such information on the microstructural and chemical features of flag leaf may have potential as markers for drought tolerance and thereby accelerate the selection and release of more drought-resistant cultivars.

Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca).

BACKGROUND: Phenotypic variation is determined by a combination of genotype, environment and their interactions. The realization that allelic diversity can be both genetic and epigenetic allows the environmental component to be further separated. Partitioning phenotypic variation observed among inbred lines with an altered epigenome can allow the epigenetic component controlling quantitative traits to be estimated. To assess the contribution of epialleles on phenotypic variation and determine the fidelity with which epialleles are inherited, we have developed a novel hypomethylated population of strawberry (2n = 2x = 14) using 5-azacytidine from which individuals with altered phenotypes can be identified, selected and characterized. RESULTS: The hypomethylated population was generated using an inbred strawberry population in the F. vesca ssp. vesca accession Hawaii 4. Analysis of whole genome sequence data from control and hypomethylated lines indicate that 5-azacytidine exposure does not increase SNP above background levels. The populations contained only Hawaii 4 alleles, removing introgression of alternate F. vesca alleles as a potential source of variation. Although genome sequencing and genetic marker data are unable to rule out 5-azacytidine induced chromosomal rearrangements as a potential source of the trait variation observed, none were detected in our survey. Quantitative trait variation focusing on flowering time and rosette diameter was scored in control and treated populations where expanded levels of variation were observed among the hypomethylated lines. Methylation sensitive molecular markers indicated that 5-azacytidine induced alterations in DNA methylation patterns and inheritance of methylation patterns were confirmed by bisulfite sequencing of targeted regions. It is possible that methylation polymorphisms might underlie or have induced genetic changes underlying the observable differences in quantitative phenotypes. CONCLUSIONS: This population developed in a uniform genetic background provides a resource for the discovery of new variation controlling quantitative traits. Genome sequence analysis indicates that 5-azacytidine did not induce point mutations and the induced variation is largely restricted to DNA methylation. Using this resource, we have identified new variation and demonstrated the inheritance of both variant trait and methylation patterns. Although direct associations remain to be determined, these data suggest epigenetic variation might be subject to selection.

Synchrotron Radiation Sheds Fresh Light on Plant Research: The Use of Powerful Techniques to Probe Structure and Composition of Plants.

While synchrotron radiation is a powerful tool in material and biomedical sciences, it is still underutilized in plant research. This mini review attempts to introduce the potential of synchrotron-based spectroscopic and imaging methods and their applications to plant sciences. Synchrotron-based Fourier transform infrared spectroscopy, X-ray absorption and fluorescence techniques, and two- and three-dimensional imaging techniques are examined. We also discuss the limitations of synchrotron-based research in plant sciences, specifically the types of plant samples that can be used. Despite limitations, the unique features of synchrotron radiation such as high brightness, polarization and pulse properties offer great advantages over conventional spectroscopic and imaging tools and enable the correlation of the structure and chemical composition of plants with biochemical function. Modern detector technologies and experimental methodologies are thus enabling plant scientists to investigate aspects of plant sciences such as ultrafast kinetics of biochemical reactions, mineral uptake, transport and accumulation, and dynamics of cell wall structure and composition during environmental stress in unprecedented ways using synchrotron beamlines. The potential for the automation of some of these synchrotron technologies and their application to plant phenotyping is also discussed.

Synchrotron based phase contrast X-ray imaging combined with FTIR spectroscopy reveals structural and biomolecular differences in spikelets play a significant role in resistance to Fusarium in wheat.

BACKGROUND: Fusarium head blight (FHB), a scab principally caused by Fusarium graminearum Schw., is a serious disease of wheat. The purpose of this study is to evaluate the potential of combining synchrotron based phase contrast X-ray imaging (PCI) with Fourier Transform mid infrared (FTIR) spectroscopy to understand the mechanisms of resistance to FHB by resistant wheat cultivars. Our hypothesis is that structural and biochemical differences between resistant and susceptible cultivars play a significant role in developing resistance to FHB. RESULTS: Synchrotron based PCI images and FTIR absorption spectra (4000-800 cm(-1)) of the floret and rachis from Fusarium-damaged and undamaged spikes of the resistant cultivar 'Sumai3', tolerant cultivar 'FL62R1', and susceptible cultivar 'Muchmore' were collected and analyzed. The PCI images show significant differences between infected and non-infected florets and rachises of different wheat cultivars. However, no pronounced difference between non-inoculated resistant and susceptible cultivar in terms of floret structures could be determined due to the complexity of the internal structures. The FTIR spectra showed significant variability between infected and non-infected floret and rachis of the wheat cultivars. The changes in absorption wavenumbers following pathogenic infection were mostly in the spectral range from 1800-800 cm(-1). The Principal Component Analysis (PCA) was also used to determine the significant chemical changes inside floret and rachis when exposed to the FHB disease stress to understand the plant response mechanism. In the floret and rachis samples, PCA of FTIR spectra revealed differences in cell wall related polysaccharides. In the florets, absorption peaks for Amide I, cellulose, hemicellulose and pectin were affected by the pathogenic fungus. In the rachis of the wheat cultivars, PCA underlines significant changes in pectin, cellulose, and hemicellulose characteristic absorption spectra. Amide II and lignin absorption peaks, persistent in the rachis of Sumai3, together with increased peak shift at 1245 cm(-1) after infection with FHB may be a marker for stress response in which the cell wall compounds related to pathways for lignification are increased. CONCLUSIONS: Synchrotron based PCI combined with FTIR spectroscopy show promising results related to FHB in wheat. The combined technique is a powerful new tool for internal visualisation and biomolecular monitoring before and during plant-microbe interactions to understand both the differences between cultivars and their different responses to disease stress.

Allium fistulosum as a novel system to investigate mechanisms of freezing resistance.

Allium fistulosum was investigated as a novel model system to examine the mechanism of freezing resistance in cold hardy plants. The 250 × 50 × 90 µm average cell size and single epidermal cell layer system allowed direct observation of endoplasmic reticulum (ER), functional group localization during acclimation, freezing and thawing on an individual cell basis in live intact tissues. Cells increased freezing resistance from an LT50 of -11°C (non-acclimated) to -25°C under 2 weeks of cold acclimation. Samples were processed using Fourier transform infrared technology (FTIR) on a synchrotron light source and a focal plane array detector. In addition, confocal fluorescent microscopy combined with a cryostage using ER selective dye of ER-Tracker allowed more detailed examination of membrane responses during freezing. Cold acclimation increased the ER volume per cell, and the freeze-induced cell deformation stopped ER streaming and ER vesiculation subsequently occurred through the breakdown in the ER network. Freeze-induced ER vesicles in cold-acclimated cells were larger and more abundant than those in non-acclimated cells. According to FTIR, the carbohydrate/ester fraction and α-helical/ß-sheet secondary structure localized in the apoplast/plasma membrane region were most visibly increased during cold acclimation. Results suggest the mechanism of cold acclimation and freezing resistance in very hardy cells may be associated with both alterations in the apoplast/plasma membrane region and the ER cryodynamics. Allium fistulosum appears to be a useful system to obtain direct evidence at both intra and extracellular levels during cold acclimation and the freezing process.

The effects of sampling and instrument orientation on LiDAR data from crop plots.

Wheat is one of the most widely consumed grains in the world and improving its yield, especially under severe climate conditions, is of great importance to world food security. Phenotyping methods can evaluate plants according to their different traits, such as yield and growth characteristics. Assessing the vertical stand structure of plants can provide valuable information about plant productivity and processes, mainly if this trait can be tracked throughout the plant's growth. Light Detection And Ranging (LiDAR) is a method capable of gathering three-dimensional data from wheat field trials and is potentially suitable for providing non-destructive, high-throughput estimations of the vertical stand structure of plants. The current study considers LiDAR and focuses on investigating the effects of sub-sampling plot data and data collection parameters on the canopy vertical profile (CVP). The CVP is a normalized, ground-referenced histogram of LiDAR point cloud data representing a plot or other spatial domain. The effects of sub-sampling of plot data, the angular field of view (FOV) of the LiDAR and LiDAR scan line orientation on the CVP were investigated. Analysis of spatial sub-sampling effects on CVP showed that at least 144000 random points (600 scan lines) or an area equivalent to three plants along the row were adequate to characterize the overall CVP of the aggregate plot. A comparison of CVPs obtained from LiDAR data for different FOV showed that CVPs varied with the angular range of the LiDAR data, with narrow ranges having a larger proportion of returns in the upper canopy and a lower proportion of returns in the lower part of the canopy. These findings will be necessary to establish minimum plot and sample sizes and compare data from studies where scan direction or field of view differ. These advancements will aid in making comparisons and inform best practices for using close-range LiDAR in phenotypic studies in crop breeding and physiology research.

Corrigendum: The effects of sampling and instrument orientation on LiDAR data from crop plots.

[This corrects the article DOI: 10.3389/fpls.2023.1087239.].

With a Little Help from My Cell Wall: Structural Modifications in Pectin May Play a Role to Overcome Both Dehydration Stress and Fungal Pathogens.

Cell wall structural modifications through pectin cross-linkages between calcium ions and/or boric acid may be key to mitigating dehydration stress and fungal pathogens. Water loss was profiled in a pure pectin system and in vivo. While calcium and boron reduced water loss in pure pectin standards, the impact on Allium species was insignificant (p > 0.05). Nevertheless, synchrotron X-ray microscopy showed the localization of exogenously applied calcium to the apoplast in the epidermal cells of Allium fistulosum. Exogenous calcium application increased viscosity and resistance to shear force in Allium fistulosum, suggesting the formation of calcium cross-linkages ("egg-box" structures). Moreover, Allium fistulosum (freezing tolerant) was also more tolerant to dehydration stress compared to Allium cepa (freezing sensitive). Furthermore, the addition of boric acid (H3BO3) to pure pectin reduced water loss and increased viscosity, which indicates the formation of RG-II dimers. The Arabidopsis boron transport mutant, bor1, expressed greater water loss and, based on the lesion area of leaf tissue, a greater susceptibility to Colletotrichum higginsianum and Botrytis cinerea. While pectin modifications in the cell wall are likely not the sole solution to dehydration and biotic stress resistance, they appear to play an important role against multiple stresses.

Diversity in Surface Microstructures of Trichomes, Epidermal Cells, and Stomata in Lentil Germplasm.

To develop crops capable of withstanding challenges posed by climate change, breeding strategies must focus on addressing multiple stresses occurring concurrently in plants. Leaf epidermal structures such as trichomes, stomata, and epidermal cells play an important role in mediating plant defense and could be essential traits that impart wide-ranging tolerance to biotic and abiotic stresses. Consequently, it is important to inform on the underlying diversity in these traits in lentil germplasm (Lens spp.). In this study, we characterized foliar microstructures of 12 genotypes belonging to seven wild and cultivated Lens species. We performed scanning electron microscopy on leaflet and pod surfaces for their qualitative characterization. For quantitative characterization, we observed surface imprints via light microscopy and quantified trichome density (TD), trichome length (TL), stomatal density (SD), epidermal cell density (ECD), and stomatal index (SI) on adaxial and abaxial leaflet surfaces for each genotype. We also assessed the heritability of trichome traits by evaluating interspecific recombinant inbred lines (RILs) derived from the cross Lens culinaris CDC Redberry × Lens tomentosus IG 72805. Comparing foliar microstructures, we found that TD and TL varied widely among cultivated and wild lentil genotypes. However, in most lentil genotypes, the adaxial leaflet surface had lower TD and longer trichomes compared to the abaxial surface. Pubescence on pods comprised five major phenotypes: no trichomes or glabrous pods, very short trichomes at low density, short trichomes at high density, medium-length trichomes at high density, and long trichomes at high density. Leaves of all species were amphistomatous, and SI, SD, and ECD were all higher on the adaxial compared to the abaxial surface. Adaxial surfaces had slightly sunken stomata, which might be an adaptive trait to conserve water. Quantifying TD and TL on the leaflets of interspecific RILs revealed transgressive segregation of these traits, suggesting that TD and TL are quantitative in nature. While taxonomic implications of this study are limited, a detailed description of agronomically relevant morphophysiological traits presented in this paper along with the mode of inheritance of trichomes may serve as a resource for scientists developing lentil adapted to concurrent biotic and abiotic stresses of the future.

Tissue specific changes in elements and organic compounds of alfalfa (Medicago sativa L.) cultivars differing in salt tolerance under salt stress.

Soil salinity is a global concern and often the primary factor contributing to land degradation, limiting crop growth and production. Alfalfa (Medicago sativa L.) is a low input high value forage legume with a wide adaptation. Examining the tissue-specific responses to salt stress will be important to understanding physiological changes of alfalfa. The responses of two alfalfa cultivars (salt tolerant 'Halo', salt intolerant 'Vernal') were studied for 12 weeks in five gradients of salt stress in a sand based hydroponic system in the greenhouse. The accumulation and localization of elements and organic compounds in different tissues of alfalfa under salt stress were evaluated using synchrotron beamlines. The pattern of chlorine accumulation for 'Halo' was: root > stem ~ leaf at 8 dSm-1, and root ~ leaf > stem at 12 dSm-1, potentially preventing toxic ion accumulation in leaf tissues. In contrast, for 'Vernal', it was leaf > stem ~ root at 8 dSm-1 and leaf > root ~ stem at 12 dSm-1. The distribution of chlorine in 'Halo' was relatively uniform in the leaf surface and vascular bundles of the stem. Amide concentration in the leaf and stem tissues was greater for 'Halo' than 'Vernal' at all salt gradients. This study determined that low ion accumulation in the shoot was a common strategy in salt tolerant alfalfa up to 8 dSm-1 of salt stress, which was then replaced by shoot tissue tolerance at 12 dSm-1.

Temperature-driven plasticity in growth cessation and dormancy development in deciduous woody plants: a working hypothesis suggesting how molecular and cellular function is affected by temperature during dormancy induction.

The role of temperature during dormancy development is being reconsidered as more research emerges demonstrating that temperature can significantly influence growth cessation and dormancy development in woody plants. However, there are seemingly contradictory responses to warm and low temperature in the literature. This research/review paper aims to address this contradiction. The impact of temperature was examined in four poplar clones and two dogwood ecotypes with contrasting dormancy induction patterns. Under short day (SD) conditions, warm night temperature (WT) strongly accelerated timing of growth cessation leading to greater dormancy development and cold hardiness in poplar hybrids. In contrast, under long day (LD) conditions, low night temperature (LT) can completely bypass the short photoperiod requirement in northern but not southern dogwood ecotypes. These findings are in fact consistent with the literature in which both coniferous and deciduous woody plant species' growth cessation, bud set or dormancy induction are accelerated by temperature. The contradictions are addressed when photoperiod and ecotypes are taken into account in which the combination of either SD/WT (northern and southern ecotypes) or LD/LT (northern ecotypes only) are separated. Photoperiod insensitive types are driven to growth cessation by LT. Also consistent is the importance of night temperature in regulating these warm and cool temperature responses. However, the physiological basis for these temperature effects remain unclear. Changes in water content, binding and mobility are factors known to be associated with dormancy induction in woody plants. These were measured using non-destructive magnetic resonance micro-imaging (MRMI) in specific regions within lateral buds of poplar under SD/WT dormancing inducing conditions. Under SD/WT, dormancy was associated with restrictions in inter- or intracellular water movement between plant cells that reduces water mobility during dormancy development. Northern ecotypes of dogwood may be more tolerant to photoinhibition under the dormancy inducing LD/LT conditions compared to southern ecotypes. In this paper, we propose the existence of two separate, but temporally connected processes that contribute to dormancy development in some deciduous woody plant: one driven by photoperiod and influenced by moderate temperatures; the other driven by abiotic stresses, such as low temperature in combination with long photoperiods. The molecular changes corresponding to these two related but distinct responses to temperature during dormancy development in woody plants remains an investigative challenge.