Rajeev Arora

Cold Acclimation in Genetically Related (Sibling) Deciduous and Evergreen Peach (Prunus persica [L.] Batsch) (II. A 60-Kilodalton Bark Protein in Cold-Acclimated Tissues of Peach Is Heat Stable and Related to the Dehydrin Family of Proteins)

In several plant species, certain cold-regulated proteins share unique properties. These proteins are (a) heat stable and (b) hydrophilic and are related to the Group 2 late embryogenesis abundant or dehydrin family of proteins. Our previous work with sibling deciduous and evergreen peach genotypes demonstrated a correlation between the level of accumulation of certain bark proteins and cold-acclimation potential of these tissues. Here we identify a 60-kD bark protein in peach (Prunus persica [L.] Batsch), PCA60 (“peach cold acclimation”), that is accumulated during cold acclimation and is heat stable. Immunological studies indicated that this protein is related to the dehydrin family of proteins and accumulates at much higher levels in the bark tissues of the deciduous genotype than in the evergreen. Amino acid composition indicated that the 60-kD protein has a compositional bias for glycine (24%), glutamic acid/glutamine (11.4%), aspartic acid/asparagine (10%), and threonine (9.6%), contains relatively low levels of aromatic amino acids (phenylalanine and tyrosine), and is rich in hydrophilic amino acids. A novel characteristic of the 60-kD cold-acclimation protein is the presence of a repeating nine-amino acid sequence. A five-amino acid stretch, which is included within this repeating motif, shares striking homology with other cold-regulated proteins and dehydrins.

Functional dissection of Hydrophilins during in vitro freeze protection

In plants, Late Embryogenesis Abundant (LEA) proteins typically accumulate in response to low water availability conditions imposed during development or by the environment. Analogous proteins in other organisms are induced when exposed to stress conditions. Most of this diverse set of proteins can be grouped according to properties such as high hydrophilicity and high content of glycine or other small amino acids in what we have termed hydrophilins. Previously, we showed that hydrophilins protect enzyme activities in vitro from low water availability effects. Here, we demonstrate that hydrophilins can also protect enzyme activities from the adverse effects induced by freeze-thaw cycles in vitro. We monitored conformational changes induced by freeze-thaw on the enzyme lactate dehydrogenase (LDH) using the fluorophore 1-anilinonaphthalene-8-sulfonate (ANS). Hydrophilin addition prevents enzyme inactivation and this effect is reflected in changes in the ANS-fluorescence levels determined for LDH. We further show that for selected plant hydrophilins, removal of certain conserved domains affects their protecting capabilities. Thus, we propose that hydrophilins, and in particular specific protein domains, have a role in protecting cell components from the adverse effects caused by low water availability such as those present during freezing conditions by preventing deleterious changes in protein secondary and tertiary structure.

Study on the decreased sugar yield in enzymatic hydrolysis of cellulosic substrate at high solid loading.

Current technology for conversion of biomass to ethanol is an enzyme-based biochemical process. In bioethanol production, achieving high sugar yield at high solid loading in enzymatic hydrolysis step is important from both technical and economic viewpoints. Enzymatic hydrolysis of cellulosic substrates is affected by many parameters, including an unexplained behavior that the glucan digestibility of substrates by cellulase decreased under high solid loadings. A comprehensive study was conducted to investigate this phenomenon by using Spezyme CP and Avicel as model cellulase and cellulose substrate, respectively. The hydrolytic properties of the cellulase under different substrate concentrations at a fixed enzyme-to-substrate ratio were characterized. The results indicate that decreased sugar yield is neither due to the loss of enzyme activity at a high substrate concentration nor due to the higher end-product inhibition. The cellulase adsorption kinetics and isotherm studies indicated that a decline in the binding capacity of cellulase may explain the long-observed but little-understood phenomenon of a lower substrate digestibility with increased substrate concentration. The mechanism how the enzyme adsorption properties changed at high substrate concentration was also discussed in the context of exploring the improvement of the cellulase-binding capacity at high substrate loading.

Comparative analysis of expressed sequence tags from cold-acclimated and non-acclimated leaves of Rhododendron catawbiense Michx

An expressed sequence tag (EST) analysis approach was undertaken to identify major genes involved in cold acclimation of Rhododendron, a broad-leaf, woody evergreen species. Two cDNA libraries were constructed, one from winter-collected (cold-acclimated, CA; leaf freezing tolerance −53°C) leaves, and the other from summer-collected (non-acclimated, NA; leaf freezing tolerance −7°C) leaves of field-grown Rhododendron catawbiense plants. A total of 862 5′-end high-quality ESTs were generated by sequencing cDNA clones from the two libraries (423 from CA and 439 from NA library). Only about 6.3% of assembled unique transcripts were shared between the libraries, suggesting remarkable differences in gene expression between CA and NA leaves. Analysis of the relative frequency at which specific cDNAs were picked from each library indicated that four genes or gene families were highly abundant in the CA library including early light-induced proteins (ELIP), dehydrins/late embryogenesis abundant proteins (LEA), cytochrome P450, and beta-amylase. Similarly, seven genes or gene families were highly abundant in the NA library and included chlorophyll a/b-binding protein, NADH dehydrogenase subunit I, plastidic aldolase, and serine:glyoxylate aminotransferase, among others. Northern blot analyses for seven selected abundant genes confirmed their preferential expression in either CA or NA leaf tissues. Our results suggest that osmotic regulation, desiccation tolerance, photoinhibition tolerance, and photosynthesis adjustment are some of the key components of cold adaptation in Rhododendron.

Intron-flanking EST–PCR markers: from genetic marker development to gene structure analysis in Rhododendron

With a long-term goal of constructing a linkage map of Rhododendron enriched with gene-specific markers, we utilized Rhododendron catawbiense ESTs for the development of high-efficiency (in terms of generating polymorphism frequency) PCR-based markers. Using the gene-sequence alignment between Rhododendron ESTs and the genomic sequences of Arabidopsis homologs, we developed ‘intron-flanking‘ EST–PCR-based primers that would anneal in conserved exon regions and amplify across the more highly diverged introns. These primers resulted in increased efficiency (61% vs. 13%; 4.7-fold) of polymorphism-detection compared with conventional EST–PCR methods, supporting the assumption that intron regions are more diverged than exons. Significantly, this study demonstrates that Arabidopsis genome database can be useful in developing gene-specific PCR-based markers for other non-model plant species for which the EST data are available but genomic sequences are not. The comparative analysis of intron sizes between Rhododendron and Arabidopsis (made possible in this study by aligning of Rhododendron ESTs with Arabidopsis genomic sequences and the sequencing of Rhododendron genomic PCR products) provides the first insight into the gene structure of Rhododendron.

Dynamics of the antioxidant system during seed osmopriming, post-priming germination, and seedling establishment in Spinach (Spinacia oleracea)

Osmopriming is a pre-sowing treatment that improves seed germination performance and stress tolerance. To understand osmopriming physiology, and its association with post-priming stress tolerance, we investigated the antioxidant system dynamics during three stages: during osmopriming, post-priming germination, and seedling establishment. Spinach seeds (Spinacia oleracea L. cv. Bloomsdale) were primed with -0.6 MPa PEG at 15°C for 8 d, and dried at room temperature for 2 d. Unprimed and primed germinating seeds/seedlings were subjected to a chilling and desiccation stresses. Seed/seedling samples were collected for antioxidant assays and germination performance and stress tolerance were evaluated. Our data indicate that: (1) during osmopriming the transition of seeds from dry to germinating state represses the antioxidant pathways (residing in dry seeds) that involve CAT and SOD enzymes but stimulates another pathway (only detectable in imbibed seeds) involving APX; (2) a renewal of antioxidant system, possibly required by seedling establishment, occurs after roughly 5 d of germination; (3) osmopriming strengthens the antioxidant system and increases seed germination potential, resulting in an increased stress tolerance in germinating seeds. Osmopriming-mediated promotive effect on stress tolerance, however, may diminish in relatively older (e.g. ~5-week) seedlings.

A 25-kDa dehydrin associated with genotype and age-dependent leaf freezing-tolerance in Rhododendron : a genetic marker for cold hardiness?

Abstract Dehydrins are plant proteins that may play a critical role in stabilizing cell functions during freezing and other dehydrative stresses. This study examines whether dehydrin expression in leaves is associated with varying levels of freezing-tolerance among F2 segregants, species, and cultivars of evergreen Rhododendron. Experiments were also conducted to determine whether physiological and chronological aging affects freezing-tolerance and dehydrin accumulation in Rhododendron leaf tissues. Our results indicate that in cold-acclimated F2 populations, levels of a 25-kDa dehydrin were closely associated with differences in leaf freezing-tolerance (LFT) among segregants. Studies of wild and cultivated plants indicated that LFT increased with both chronological age and developmental phase-change (juvenile to mature plants) and that this trend was accompanied by increased accumulation of the 25-kDa dehydrin. It is suggested that presence or absence of the 25-kDa dehydrin could serve as a genetic marker to distinguish between super cold-hardy and less cold-hardy rhododendron genotypes. Similarly, the relative level of this protein within a genotype can serve as a physiological indicator of freezing-tolerance status under a range of phenological (acclimation) or developmental (age) conditions.

Major differences observed in transcript profiles of blueberry during cold acclimation under field and cold room conditions

Our laboratory has been working toward increasing our understanding of the genetic control of cold hardiness in blueberry (Vaccinium section Cyanococcus) to ultimately use this information to develop more cold hardy cultivars for the industry. Here, we report using cDNA microarrays to monitor changes in gene expression at multiple times during cold acclimation under field and cold room conditions. Microarrays contained over 2,500 cDNA inserts, approximately half of which had been picked and single-pass sequenced from each of two cDNA libraries that were constructed from cold acclimated floral buds and non-acclimated floral buds of the fairly cold hardy cv. Bluecrop (Vaccinium corymbosum L.). Two biological samples were examined at each time point. Microarray data were analyzed statistically using t tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). Interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Many of the genes induced only under cold room conditions could be divided into three major types: (1) genes associated with stress tolerance; (2) those that encode glycolytic and TCA cycle enzymes, and (3) those associated with protein synthesis machinery. A few of the genes induced only under field conditions appear to be related to light stress. Possible explanations for these differences are discussed in physiological context. Although many similarities exist in how plants respond during cold acclimation in the cold room and in the field environment, there are major differences suggesting caution should be taken in interpreting results based only on artificial, cold room conditions.

Changes in carbohydrates, ABA and bark proteins during seasonal cold acclimation and deacclimation in Hydrangea species differing in cold hardiness

Cold injury is frequently seen in the commercially important shrub Hydrangea macrophylla but not in Hydrangea paniculata. Cold acclimation and deacclimation and associated physiological adaptations were investigated from late September 2006 to early May 2007 in stems of field-grown H. macrophylla ssp. macrophylla (Thunb.) Ser. cv. Blaumeise and H. paniculata Sieb. cv. Kyushu. Acclimation and deacclimation appeared approximately synchronized in the two species, but they differed significantly in levels of mid-winter cold hardiness, rates of acclimation and deacclimation and physiological traits conferring tolerance to freezing conditions. Accumulation patterns of sucrose and raffinose in stems paralleled fluctuations in cold hardiness in both species, but H. macrophylla additionally accumulated glucose and fructose during winter, indicating species-specific differences in carbohydrate metabolism. Protein profiles differed between H. macrophylla and H. paniculata, but distinct seasonal patterns associated with winter acclimation were observed in both species. In H. paniculata concurrent increases in xylem sap abscisic acid (ABA) concentrations ([ABA](xylem)) and freezing tolerance suggests an involvement of ABA in cold acclimation. In contrast, ABA from the root system was seemingly not involved in cold acclimation in H. macrophylla, suggesting that species-specific differences in cold hardiness may be related to differences in [ABA](xylem). In both species a significant increase in stem freezing tolerance appeared long after growth ceased, suggesting that cold acclimation is more regulated by temperature than by photoperiod.

Genetic analysis of freezing tolerance in blueberry (Vaccinium section Cyanococcus)

Abstract An understanding of the genetic control of freezing tolerance (FT) in woody perennials is important for the effective selection and development of plants with a broader climatic adaptation. This study was undertaken to examine the inheritance and gene action of FT in segregating populations of a woody perennial blueberry (Vaccinium, section Cyanococcus). Two backcross populations were derived from interspecific hybrids of the diploid species Vaccinium darrowi andVaccinium caesariense, which are widely divergent in their FT. The bud FTs of uniformly cold acclimated plants of parental, F1, and two backcross populations were evaluated with a laboratory controlled freeze-thaw regime, followed by a visual assessment of injury. FT (LT50) was defined as the temperature causing 50% of the flower buds to be injured. Data indicate that the two parents were homozygous for genes for low or high FT. Freezing-tolerance values of the parental and F1 populations indicate that freeze-sensitivity is a partially dominant trait. Results from reciprocal crosses revealed that there was no significant maternal influence on freezing tolerance. Parental phenotypes were fully recovered in 40–42 plants of each testcross population, suggesting that FT is determined by relatively few genes. The degree of dominance and an analysis of generation means revealed that FT in blueberry is controlled largely by additive gene effects and, to a lesser degree, by dominance gene effects. Testing of various genetic models indicated that FT inheritance can be adequately explained by a simple additive-dominance model; however, two epistatic models involving additive-additive and dominance-dominance interactions also fit the data.