Summary
Experimental studies on CO2 assimilation of mesophytic C3 plants in relation to relative water content (RWC) are discussed. Decreasing RWC slows the actual rate of photosynthetic CO2 assimilation (A) and decreases the potential rate (Apot). Generally, as RWC falls from c. 100 to c. 75%, the stomatal conductance (gs) decreases, and with it A. However, there are two general types of relation of Apot to RWC, which are called Type 1 and Type 2. Type 1 has two main phases. As RWC decreases from 100 to c. 75%, Apot is unaffected, but decreasing stomatal conductance (gs) results in smaller A, and lower CO2 concentration inside the leaf (Ci) and in the chloroplast (Cc), the latter falling possibly to the compensation point. Down-regulation of electron transport occurs by energy quenching mechanisms, and changes in carbohydrate and nitrogen metabolism are considered acclimatory, caused by low Ci and reversible by elevated CO2. Below 75% RWC, there is metabolic inhibition of Apot, inhibition of A then being partly (but progressively less) reversible by elevated CO2; gs regulates A progressively less, and Ci and CO2 compensation point, Γ rise. It is suggested that this is the true stress phase, where the decrease in Apot is caused by decreased ATP synthesis and a consequent decreased synthesis of RuBP. In the Type 2 response, Apot decreases progressively at RWC 100 to 75%, with A being progressively less restored to the unstressed value by elevated CO2. Decreased gs leads to a lower Ci and Cc but they probably do not reach compensation point: gs becomes progressively less important and metabolic limitations more important as RWC falls. The primary effect of low RWC on Apot is most probably caused by limited RuBP synthesis, as a result of decreased ATP synthesis, either through inhibition of Coupling Factor activity or amount due to increased ion concentration. Carbohydrate synthesis and accumulation decrease. Type 2 response is considered equivalent to Type 1 at RWC below c. 75%, with Apot inhibited by limited ATP and RuBP synthesis, respiratory metabolism dominates and Ci and Γ rise. The importance of inhibited ATP synthesis as a primary cause of decreasing Apot is discussed. Factors determining the Type 1 and Type 2 responses are unknown. Electron transport is maintained (but down-regulated) in Types 1 and 2 over a wide range of RWC, and a large reduced/oxidized adenylate ratio results. Metabolic imbalance results in amino acid accumulation and decreased and altered protein synthesis. These conditions profoundly affect cell functions and ultimately cause cell death. Type 1 and 2 responses may reflect differences in gs and in sensitivity of metabolism to decreasing RWC.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.0016-8025.2001.00814.x

Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in many places in living cells and at an increased rate during biotic or abiotic stress. ROS and RNS participate in signal transduction, but also modify cellular components and cause dam- age. We first look at the most common ROS and their properties. We then consider the ways in which the cell can regulate their pro- duction and removal. We critically assess current knowledge about modifications of polyunsaturated fatty acids (PUFAs), DNA, carbo- hydrates, and proteins and illustrate this knowledge with case stories wherever possible. Some oxidative breakdown products, e.g., from PUFA, can cause secondary damage. Other oxidation products are secondary signaling molecules. We consider the fate of the modi- fied components, the energetic costs to the cell of replacing such components, as well as strategies to minimize transfer of oxidatively damaged components to the next generation.

Oxidative Modifications to Cellular Components in Plants

The production of reactive oxygen species (ROS), such as O2- and H2O2, is an unavoidable consequence of aerobic metabolism. In plant cells the mitochondrial electron transport chain (ETC) is a major site of ROS production. In addition to complexes I-IV, the plant mitochondrial ETC contains a non-proton-pumping alternative oxidase as well as two rotenone-insensitive, non-proton-pumping NAD(P)H dehydrogenases on each side of the inner membrane: NDex on the outer surface and NDin on the inner surface. Because of their dependence on Ca2+, the two NDex may be active only when the plant cell is stressed. Complex I is the main enzyme oxidizing NADH under normal conditions and is also a major site of ROS production, together with complex III. The alternative oxidase and possibly NDin(NADH) function to limit mitochondrial ROS production by keeping the ETC relatively oxidized. Several enzymes are found in the matrix that, together with small antioxidants such as glutathione, help remove ROS. The antioxidants are kept in a reduced state by matrix NADPH produced by NADP-isocitrate dehydrogenase and non-proton-pumping transhydrogenase activities. When these defenses are overwhelmed, as occurs during both biotic and abiotic stress, the mitochondria are damaged by oxidative stress.

PLANT MITOCHONDRIA AND OXIDATIVE STRESS: Electron Transport, NADPH Turnover, and Metabolism of Reactive Oxygen Species.

A vast number of plant pathogens from viroids of a few hundred nucleotides to higher plants cause diseases in our crops. Their effects range from mild symptoms to catastrophes in which large areas planted to food crops are destroyed. Catastrophic plant disease exacerbates the current deficit of food supply in which at least 800 million people are inadequately fed. Plant pathogens are difficult to control because their populations are variable in time, space, and genotype. Most insidiously, they evolve, often overcoming the resistance that may have been the hard-won achievement of the plant breeder. In order to combat the losses they cause, it is necessary to define the problem and seek remedies. At the biological level, the requirements are for the speedy and accurate identification of the causal organism, accurate estimates of the severity of disease and its effect on yield, and identification of its virulence mechanisms. Disease may then be minimized by the reduction of the pathogen's inoculum, inhibition of its virulence mechanisms, and promotion of genetic diversity in the crop. Conventional plant breeding for resistance has an important role to play that can now be facilitated by marker-assisted selection. There is also a role for transgenic modification with genes that confer resistance. At the political level, there is a need to acknowledge that plant diseases threaten our food supplies and to devote adequate resources to their control.

Plant Disease: A Threat to Global Food Security

The INK4a-ARF locus encodes two proteins, p16INK4a and p19ARF, that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively. Disruption of this locus by deletions or point mutations is a common event in human cancer, perhaps second only to the loss of p53. Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we determined that mouse p19ARF can interact directly with p53, as well as with the p53 regulator mdm2. ARF can bind p53-DNA complexes, and it depends upon functional p53 to transcriptionally induce mdm2 and the cyclin-dependent kinase inhibitor p21Cip1, and to arrest cell proliferation. Binding of p19ARF to p53 requires the ARF N-terminal domain (amino acids 1–62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19ARF in wild type or ARF-null mouse embryo fibroblasts increases the half-life of p53 from 15 to ≈75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra-physiologic levels after introduction into ARF-null NIH 3T3 cells or mouse embryo fibroblasts, the p53 protein is handicapped in inducing this checkpoint response. In this setting, reintroduction of p19ARF restores p53’s ability to induce p21Cip1 and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53-dependent function through an additional mechanism.

https://www.pnas.org/content/pnas/95/14/8292.full.pdf

Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2

We have previously regenerated tobacco plants from fused protoplasts isolated from two varieties of Nicotiana tabacum. The parent cells had distinct morphological nuclear and cytoplasmic markers, enabling us to recognise, among the whole regenerated plants, those with a single parental nucleus and a hybrid cytoplasm produced by the mixing of the two parental cytoplasms1. Genetic analysis of their progeny has confirmed that the phenotypes of these cytoplasmic hybrids (or cybrids) are stable and maternally inherited. Analysis with restriction endonuclease has shown that only one or the other parental chloroplast DNA is present in the progeny of the cybrids2. We report here, however, that the mitochondrial (mt) DNAs of cybrids are different from those of the parents and from the mixture of the two. The new DNAs result from mitochondrial recombination.

Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion

We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CR IS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, Ch IS I and II display similar alterations, Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins, Mitochondria isolated from CMS leaves showed altered respiration, Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced, The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity, Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I, Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS. and higher amounts of enzyme were evidenced by immunodetection.

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants

Protoplast fusion makes possible the fusion of two different cytoplasms, allowing genetical analysis of cytoplasmic factors. Two varieties of Nicotiana tabacum differing by their cytoplasms have been used. Techne, the first variety, obtained by an interspecific cross between N. debneyi (female) and N. tabacum (male) is characterized by the nuclear tabacum genome inside the debneyi cytoplasm. Techne plants present abnormal flowers with cytoplasmic male sterility (cytoplasmic marker) and sessile leave (nuclear marker). Techne leaf protoplasts were fused with leaf protoplasts of N. tabacum var. Samsun (or Xanthi). The last variety is characterized by petiolated leaves and normal flowers, because it possesses the nuclear tabacum genome associated with the tabacum cytoplasm. The nuclear marker (leaf shape) and the cytoplasmic one (flower shape inducing male sterility or fertility) have been used to distinguish among the whole regenerated plants the somatic nuclear hybrids and the cytoplasmic hybrids (cybrids) displaying the nuclear phenotype of one of the two parents associated with a modified flower type, intermediate between the parental ones.

Morphological characteristics and chloroplast DNA distribution in different cytoplasmic parasexual hybrids of Nicotiana tabacum

THE physicochemical properties of mitochondrial (mt)-DNA of higher plants have been reported1–4. mt-DNAs occur as closed circles with molecular weights of (60–70) × 106 and it is likely that each mitochondria contains several of these circles. mt-DNAs isolated from different higher plants exhibit similar physicochemical parameters: all mt-DNAs so far analysed1–5 in higher plants band at a unique buoyant density of 1.706 g cm−3 in neutral CsCl gradients and the few published melting points and reassociation kinetics are not significantly different2–4. Not enough values have been reported to state on possible differences in the contour length measured by electron microscopy. Thus, mt-DNAs isolated from different higher plants cannot be specifically identified by conventional physicochemical techniques. We have recently reported that the analysis of restriction nuclease digests by gel electrophoresis provides a very sensitive test to identify specifically chloroplastic (cp)-DNAs isolated from different plant genera and species6. We show here that this technique also allows the characterisation and the specific identification of mt-DNAs isolated from various higher plants. We used EcoRI specific cleavage to evidence the general occurrence of heterogeneous population of mt-DNA molecules within an higher plant and to detect clear differences between mt-DNAs extracted from normal and cytoplasmic male-sterile wheat.

Fernand Vedel

Papers

Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants

Morphological characteristics and chloroplast DNA distribution in different cytoplasmic parasexual hybrids of Nicotiana tabacum

Heterogeneous population of mitochondrial DNA molecules in higher plants

Study of wheat phylogeny by coRI analysis of chloroplastic and mitochondrial DNAs