Università Cattolica del Sacro Cuore
Chloroplast morphology, pigment content and fluorescence parameters in virescent mutants --Marocco, A Chlorophyll-deficient tissue is a frequent symptom in plants when they experience low temperature. These symptoms also arise in mutated populations of plants. The virescent character is a common genetic variant in pigmentation of higher plants for which a large number is known in maize. It is generally accepted that expression of this character is influenced by temperature sensitivity (reviewed by King, 1991 The Genetic Basis of Plant Physiological Processes, Oxford Univ. Press, 151-224). Since the cloning of genes affecting chloroplast development at low temperature may help application oriented activities in the field of temperature tolerance in maize, we developed gene tagging experiments for the Virescent genes of maize using the Activator-Dissociation transposable elements (Cerioli et al., 1995 Genet. Res. 66: 203-212). I have now examined the phenotypic expression of virescent mutations by analysing the chloroplast morphology, the pigment content and the fluorescence parameters in nine mutants grown at low temperature.

The v1, v2, v3, v4, v13, v16, v18, v19 and v26 mutants employed in this study are derived from progenitor stocks obtained from the Maize Genetics Cooperation Stock Center, University of Illinois, Urbana. All mutants are in the Oh43 background. Seeds were grown in a controlled environment chamber at 15 C under continuous light (80 Wm-2).

Electron microscope inspection of the virescent leaf phenotype at the cellular level showed that cells in the leaf tissue contain few chloroplasts (Table 1), and these did not appear to be fully developed. Plastids contain poorly organised and aberrant thylakoids; the organelles generally are two-thirds the size of fully developed chloroplasts. The ungreened virescent leaves contained a variety of aberrant chloroplasts. The plastid types ranged from those with little internal structure on loosely constructed prolamellar bodies to those with nearly normal morphology. The distinctly abnormal plastids predominated in v2, v4 and v16.

The level of photosynthetic pigments is severely reduced (Table 1). The virescent mutants were extremely deficient in chlorophyll, which was to near-normal level with Oh43 genotype. The v1, v2, v3, v19 and v26 leaves were relatively less deficient in chlorophyll b than in chlorophyll a, resulting in lower chlorophyll a/b ratios than for normal leaves. Leaves of v4 and v13 exhibited a substantial increase in chlorophyll a relative to the chlorophyll b content. Changes in the proportion of luteins and xanthophylls per unit weight in normal and virescent leaves were measured. Leaves of v1, v19 and v13 exhibited a decrease by 44%, 40% and 16%, respectively, in the lutein content compared to wild type. The lutein contents of v2, v3, v4, v16, v18 and v26 were 31% below the wild type. The levels of xanthophylls were higher in leaves of v1, v13 and v19 as compared to wild type. The mutations led to an increase in the relative levels from 53% to 70%. A strong reduction in the xanthophyll contents by more than 64% was observed in v2, v3, v4, v16, v18 and v26 mutants compared to normal leaves.

Fluorescence measurements were performed with a PAM 101 fluorimeter (Heinz Walz, Effeltrich, Germany). From an analysis of the fluorescence quenching parameters in the green tips of leaves, it is shown that all mutants possess a functioning, fully reversible, non-photochemical quenching mechanism (Table 1). This is most developed in the v13, v18 and v19 mutants. These three mutants also have a relatively high primary photochemical yield for photosystem II and a functioning photosystem I, as indicated by the high photochemical quenching capacity. Together the chlorotic phenotype and the molecular identification will provide a foundation to investigate the pathway for this mechanism of cold susceptibility.

The various virescent mutations are characterised by different stages of chloroplast development. I hypothesize a model where V2, V4 and V16 loci are located upstream, followed by V1, V3 and V26. Mutations in V13, V18 and V19 loci may control the downstream pathway. The leaves of ungreened virescents contain a reduced amount of total chorophyll. The relatively greater deficiency in chlorophyll a reduces the chlorophyll a/b ratio below normal in most of the mutations. This difference can not be regarded as a distinctive characteristic of all virescent mutants as reported for the v18 mutant (Chollet and Paolillo, 1972 Z. Pflanzenphysiol. 68: 30-44). In fact, the ratio is considerably higher in v4 and v13 leaves. The total content of carotenoids was affected to a much lesser extent, if at all, by the mutations. It seems, therefore, that mutant leaves attempt to protect themselves against damage from excessive light by increasing the carotenoids/chlorophyll a+b ratio (Haldimann et al., 1995 Physiol. Plant. 95:409-414). In this context it is interesting that all mutants possess a significant, reversible, non-photochemical quenching capacity. From the point of view of the photosynthetic characteristics determined by PAM fluorescence measurements, the most interesting mutants are v13, v18 and v19. These mutants not only have a relatively high PSII quantum efficiency for primary energy conversion when grown under cold stress conditions, but evidently also have an actively functioning PSI, as indicated by the high values for photochemical quenching. In addition, these three mutants also possess the most effective non-photochemical quenching mechanism(s), which is thought to provide protection against excess photon absorption by PSII. Even more important is the finding that the mutations induced important changes in the photosynthetic quantum conversion. Photosystem II appears to be correctly assembled in the v13, v18 and v19 mutants because the Fv/Fm, fp, fn and ftot were higher than wild type. In these mutants the protections against low temperature and excessive light is ensured by the fp and fn processes.

Table 1. Plastid measurements, chlorophyll content and in vivo chlorophyll fluorescence in normal and virescent leaves grown at 15 C. Data are means of 5-7 replicates, each representing a different plant ± SE.
Genotype Plastid numbera Plastid size (mm) Chlorophyll a+b Fv/Fm
v1 5.0±1.0 3.3 ± 0.2 x 2.4 ± 0.3 44.0±1.0 0.32
v2 4.0±0.6 5.5 0.8 ± x 2.5 ± 0.4 10.0±0.1 0.12
v3 4.7±0.8 4.0 ± 0.7 x 2.0 ± 0.0 37.7±0.7 n.d.
v4 3.5±0.5 4.0 ± 0.7 x 2.0 ± 0.0 17.7±0.4 n.d.
v13 5.7±1.0 6.8 ± 0.8 x 4.3 ± 0.3 102.0±1.7 0.67
v16 3.9±0.5 4.0 ± 0.5 x 2.5 ± 0.2 traces 0.09
v18 3.0±0.0 6.0 ± 1.0 x 2.5 ± 0.3 7.8±0.1 0.52
v19 4.4±0.5 6.0 ± 0.6 x 3.0 ± 0.4 67.0±0.8 0.62
V26 3.9±0.5 6.0 ± 0.5 x 3.0 ± 0.1 13.3±0.1 0.35
Oh43 6.1±0.7 4.5 ± 0.4 x 3.0 ± 0.2 3,000±9.4 0.81
aEach entry represents the average of measurements obtained by analysing 20 cells.

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