SummaryTransgenic alfalfa root cultures expressing sense and antisense barley hemoglobin transcripts were examined under varying levels of atmospheric oxygen. Root cultures overexpressing the hemoglobin gene (Hb ) maintained root growth when placed under 3% oxygen, whereas control cultures or cultures underexpressing hemoglobin (Hb À ) experienced 30±70% declines in growth under the same conditions. ATP levels and ATP/ADP ratios for Hb lines did not signi®cantly differ in 40 and 3% oxygen, whereas the ATP levels and ATP/ADP ratios in control and Hb À lines were signi®cantly lower under 3% oxygen. Large increases in the production of nitric oxide (NO) were measured in root cultures grown under hypoxic conditions compared to aerobic conditions. The amount of NO accumulated in an Hb À line was 2.5-fold higher than that in the Hb line. Treatment of transgenic root lines under 40% oxygen with NO resulted in signi®cant declines in the ATP levels and ATP/ADP ratio of an Hb À line and the control line, with no signi®cant change in an Hb line. The root cell structure of an Hb À line showed evidence of cell breakdown under hypoxic growth, whereas an Hb line had no evidence of cell breakdown under similar growth conditions. These results lead us to hypothesize that NO is involved in the response of plants to hypoxia and that hemoglobin modulates the levels of NO in the hypoxic cell.

In common with other zwitterionic quaternary ammonium compounds (QACs), glycine betaine acts as an osmoprotectant in plants, bacteria, and animals, with its accumulation in the cytoplasm reducing adverse effects of salinity and drought. For this reason, the glycine betaine biosynthesis pathway has become a target for genetic engineering of stress tolerance in crop plants. Besides glycine betaine, several other QAC osmoprotectants have been reported to accumulate among flowering plants, although little is known about their distribution, evolution, or adaptive value. We show here that various taxa of the highly stress-tolerant family Plumbaginaceae have evolved four QACs, which supplement or replace glycine betaine-namely, choline 0-sulfate and the betaines of (-alanine, proline, and hydroxyproline. Evidence from bacterial bioassays demonstrates that these QACs function no better than glycine betaine as osmoprotectants. However, the distribution of QACs among diverse members of the Plumbaginaceae adapted to different types of habitat indicates that different QACs could have selective advantages in particular stress environments. Specifically, choline 0-sulfate can function in sulfate detoxification as well as in osmoprotection, 13-alanine betaine may be superior to glycine betaine in hypoxic saline conditions, and proline-derived betaines may be beneficial in chronically dry environments. We conclude that the evolution of osmoprotectant diversity within the Plumbaginaceae suggests additional possibilities to explore in the metabolic engineering of stress tolerance in crops.Abiotic stresses such as drought and salinity are the major constraints to crop yield (1), and more sources of genes for tolerance to them are needed (2-4). When such genes occur in crops or in closely related wild species, they can be exploited by traditional breeding techniques (3, 4). A much wider potential pool of genes is now available: genetic engineering makes it possible to use any organism as a source of simple adaptations to abiotic stresses (5, 6). This has led to much interest in stress adaptations that may be controlled by one or a few genes.One such adaptation to dry and saline conditions is the accumulation of osmoprotectants (7). Unlike most solutes, osmoprotectants stabilize proteins and membranes when present at high concentrations and so can be used to raise cytoplasmic osmotic pressure in stressed cells without deleterious effects (7, 8). Betaines and other zwitterionic quaternary ammonium compounds (QACs) are very effective osmoprotectants, and several occur in diverse taxa of flowering plants (Table 1 and ref. 9). Glycine betaine is the most widespread of these; it is the only one for which biosynthetic enzymes and genes have been isolated (10, 11) and the onlyThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. one so far targeted for metabolic engineering (1...

It is now known that there are several classes of haemoglobins in plants. A specialized class of haemoglobins, symbiotic haemoglobins, were discovered 62 years ago and are found only in nodules of plants capable of symbiotic nitrogen fixation. Plant haemoglobins, with properties distinct from symbiotic haemoglobins were discovered 18 years ago and are now believed to exist throughout the plant kingdom. They are expressed in different organs and tissues of both dicot and monocot plants. They are induced by hypoxic stress and by oversupply of certain nutrients. Most recently, truncated haemoglobins have been shown to also exist in plants. While hypoxic stress-induced haemoglobins are widespread in the plant kingdom, their function has not been elucidated. This review discusses the recent findings regarding the function of these haemoglobins in relation to adaptation to hypoxia in plants. We propose that nitric oxide is an important metabolite in hypoxic plant cells and that at least one of the functions of hypoxic stress-induced haemoglobins is to modulate nitric oxide levels in the cell.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are present at low and controlled levels under normal conditions. These reactive molecules can increase to high levels under various biotic and abiotic conditions, resulting in perturbation of the cellular redox state that can ultimately lead to oxidative or nitrosative stress. In this review, we analyze the various effects that result from alterations of redox homeostasis on plant glycolytic pathway and tricarboxylic acid (TCA) cycle. Most documented modifications caused by ROS or RNS are due to the presence of redox-sensitive cysteine thiol groups in proteins. Redox modifications include Cys oxidation, disulfide bond formation, S -glutathionylation, S -nitrosylation, and S -sulfhydration. A growing number of proteomic surveys and biochemical studies document the occurrence of ROS- or RNS-mediated modification in enzymes of glycolysis and the TCA cycle. In a few cases, these modifications have been shown to affect enzyme activity, suggesting an operational regulatory mechanism in vivo . Further changes induced by oxidative stress conditions include the proposed redox-dependent modifications in the subcellular distribution of a putative redox sensor, NAD-glyceraldehyde-3P dehydrogenase and the micro-compartmentation of cytosolic glycolytic enzymes. Data from the literature indicate that oxidative stress may induce complex changes in metabolite pools in central carbon metabolism. This information is discussed in the context of our understanding of plant metabolic response to oxidative stress.

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