Jean-Louis Prioul

Mitochondria-Driven Changes in Leaf NAD Status Exert a Crucial Influence on the Control of Nitrate Assimilation and the Integration of Carbon and Nitrogen Metabolism

The Nicotiana sylvestris mutant, CMS, lacks the mitochondrial gene nad7 and functional complex I, and respires using low-affinity NADH (alternative) mitochondrial dehydrogenases. Here, we show that this adjustment of respiratory pathways is associated with a profound modification of foliar carbon-nitrogen balance. CMS leaves are characterized by abundant amino acids compared to either wild-type plants or CMS in which complex I function has been restored by nuclear transformation with the nad7 cDNA. The metabolite profile of CMS leaves is enriched in amino acids with low carbon/nitrogen and depleted in starch and 2-oxoglutarate. Deficiency in 2-oxoglutarate occurred despite increased citrate and malate and higher capacity of key anaplerotic enzymes, notably the mitochondrial NAD-dependent isocitrate dehydrogenase. The accumulation of nitrogen-rich amino acids was not accompanied by increased expression of enzymes involved in nitrogen assimilation. Partitioning of 15N-nitrate into soluble amines was enhanced in CMS leaf discs compared to wild-type discs, especially in the dark. Analysis of pyridine nucleotides showed that both NAD and NADH were increased by 2-fold in CMS leaves. The growth retardation of CMS relative to the wild type was highly dependent on photoperiod, but at all photoperiod regimes the link between high contents of amino acids and NADH was observed. Together, the data provide strong evidence that (1) NADH availability is a critical factor in influencing the rate of nitrate assimilation and that (2) NAD status plays a crucial role in coordinating ammonia assimilation with the anaplerotic production of carbon skeletons.

Expression of ADP-Glucose Pyrophosphorylase in Maize (Zea mays L.) Grain and Source Leaf during Grain Filling

The time course of ADP-glucose pyrophosphorylase activity and of starch accumulation rate measured in grain, from pollination to maturity, in Zea mays L. plants grown outdoors, was coincident for 2 years. No such correlation was observed in the adjacent leaf, which, furthermore, presented large year-to-year differences in starch accumulation pattern. Analysis of the expression of ADP-glucose synthase at the protein level, using antibodies directed against the Bt2 or Sh2 subunits, established that the variation of activity in the grain was explained by parallel changes in the content of both subunits. The cDNA for Bt2 and Sh2 subunits were used as probes to quantify the corresponding messenger. In grain, the time course of Bt2 and Sh2 mRNA accumulation anticipated, with a similar pattern, the specific peptide variations, which suggests a transcriptional control of expression. By contrast, the control of leaf activity by protein content was less obvious than in the grain, and changes in leaf enzyme specific activity were suggested during the first 20 d after pollination. A clone homologous to the grain Bt2 subunit cDNA was isolated from a maize leaf cDNA library, and a sequence comparison showed that the leaf clone (L2) was a partial cDNA representing one-third of the mature peptide. A 97% homology was observed between Bt2 and L2 in their coding region, but homology was poor in the 3[prime] noncoding border. This result demonstrates that Bt2 and L2 arise from different genes presenting a tissue-specific expression pattern and provides an explanation for the earlier reported differences between leaf and grain in the size of peptide and mRNA for the Bt2-homologous subunit.

Genetic dissection of the relationship between carbon metabolism and early growth in maize, with emphasis on key-enzyme loci

The determinism of carbon metabolism traits during early growth in maize has been investigated using a marker-based quantitative genetics approach. In addition to growth traits, concentration of carbohydrates and activity of four key enzymes of their metabolism (sucrose phosphate synthase, ADP-glucose pyrophosphorylase, invertases and sucrose synthase) have been measured in leaves of individuals of a recombinant inbred line population. Using more than 100 RFLP markers, quantitative trait loci (QTLs) were mapped for each biochemical and developmental trait. Causal relationships, suggested by previous physiological studies, were reinforced by common locations of QTLs for different traits. Thus, the strong correlation between growth rate and invertase activity, which may reflect sink organ strength, could be explained to a large extent by a single region of chromosome 8. Moreover, some of the structural genes of the enzymes mapped to regions with QTLs affecting the activity of the encoded enzyme and/or concentration of its product, and sometimes growth traits. These results emphasize the possible role of the polymorphism of key-enzyme genes in physiological processes, and hence in maize growth.

Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases.

Two maize putative cell-wall invertase genes (Incw3 and Incw4) have been isolated by screening a genomic DNA library (Zea mays L. W22) using the cDNA probes encoding the two maize cell-wall invertases Incw1 and Incw2. The Incw3 and Incw4 genes contain six exons/five introns and five exons/four introns, respectively. The protein sequences deduced from both genes revealed a beta-fructosidase motif and a cysteine catalytic site known to be conserved in invertase genes. A detailed analysis of the protein and nucleotide sequences provides evidence that the Incw3 and the Incw4 genes encode putative cell-wall invertases. Furthermore, the isoelectric point deduced from the INCW4 protein sequence suggested that the Incw4 gene may encode a unique type of cell-wall invertase unbound in the apoplast. Gene expression studies using RT-PCR and in-situ RT-PCR hybridization showed that the Incw3 expression is organ/tissue-specific and developmentally regulated. In contrast, the Incw4 gene is constitutively expressed in all vegetative and reproductive tissues tested.

Ivr2, a candidate gene for a QTL of vacuolar invertase activity in maize leaves. Gene-specific expression under water stress.

Water shortage produced an early and large stimulation of acid- soluble invertase activity in adult maize leaves whereas cell wall invertase activity remained constant. This response was closely related to the mRNA level for only one of the invertase gene (Ivr2), encoding a vacuolar isoform. In parallel, four quantitative trait loci (QTLs) were detected for invertase activity under control and nine under stressful conditions. One QTL in control and one in stressed plants was located near to the lvr2 gene on chromosome 5. Other QTLs for invertase activity were found close to carbohydrate QTLs; some of them formed stress ‘clusters’.

Isolation, Characterization and Expression Analyses of Two Cell Wall Invertase Genes in Maize

Summary Acid invertases are glycoproteins that catalyze the hydrolysis of sucrose to glucose and fructose and are associated with metabolic sink tissues in a variety of plant species. Acid invertases are divided into cell wall-bound invertases (INCW) and soluble invertases based on their location in the cell. We describe here the isolation and characterization of two cell wall invertase cDNA ( Incw 1 and Incw 2) and genomic clones. Since the deduced amino acid sequences of Incw 1 and Incw 2 clones are more similar to carrot cell wall invertases than they are to maize soluble invertase, we conclude Incw 1 and Incw 2 represent cell wallbound invertases. Both genomic clones have six introns and seven exons, typical of most other acid invertase genes. Incw 1 mRNA is present in cell suspension culture, etiolated shoots, roots and, at much reduced steady state levels, in developing endosperm. In contrast, Incw 2 mRNA is present in shoots and developing endosperm, but lacking in roots and the miniature 1 ( mn 1-1) mutant endosperm. In situ hybridization studies show that the Incw 2 mRNA is confined to the basal endosperm transfer cells in a developing kernel.

Effect of nitrate on water transfer across roots of nitrogen pre-starved maize seedlings

The addition of 10 mM KNO3 to the solution bathing the roots of young nitrogen-starved seedlings of Zea mays L. enhanced root water transfer within 15 h, compared with 10 mM KCl addition. The free exudation flux was 2.2–3.9 times higher in excised KNO3-treated roots than in KCl-treated ones. Cryo-osmometry data for xylem sap suggested that, compared with chloride, nitrate treatment increased the steady solute flux into the xylem, but did not modify the osmotic concentration of sap. Root growth was not significantly modified by nitrate within 15 h. Root hydraulic conductances were measured by using either hydrostatic-pressure or osmotic-gradient methods. During hydrostatic experiments, the conductance (kp), which is thought to refer mainly to the apoplasmic pathway, was 1.6 times larger in KNO3-than in KCl-treated plants. From experiments in which polyethylene glycol (PEG) 8000 was used as external osmolyte, osmotic conductances (ks) were found to be smaller by 5–20 times than kp for the two kinds of plants. The KCl-treated roots were characterized by a low ks which was the same for influx or efflux of water. By contrast, KNO3-treated roots exhibited two distinct conductances ks1 and ks2, indicating that influx of water was easier than efflux when the water flow was driven by the osmotic pressure gradient. Infiltration of roots with KNO3 solution supported the idea that nitrate might enhance the efficiency of the cell-to-cell pathway. The low ks value of KCl-treated roots and the existence of two contrasting ks values (ks1 and ks2) for KNO3-treated roots are discussed in terms of reversible closing of water channels.

Acclimation of adult Lolium multiflorum leaves to changes in irradiance: effect on leaf photosynthesis and chloroplast ultrastructure

Summary Photosynthetic and morphological features were examined in fully expanded leaves of plants transferred either from low to high or high to low light conditions and compared to control leaves maintained under constant high or low light. In both control conditions, net maximum photosynthetic rate, stomatal and intracellular conductances declined about one week following full leaf expansion suggesting the onset of leaf senescence. Plants transferred from low to high light at the time of full expansion of experimental leaf (3rd) showed an increase in all photosynthetic parameters within 2–3 days, a plateau (5–7 days) and then a decline. In the reciprocal transfer from high to low light, a dramatic decrease in photosynthetic characteristics occurred after 4 days, progressively levelling off to control low light values. Low-light control chloroplasts showed a typical dense thylakoid system versus a more reduced network in the high light counterpart. A transfer from low to high light induced a reduction in the relative lamellar content thus readjusting to a typical high-light plastid morphology, while in the reciprocal transfer renewed thylakoid growth or rearrangement led to a dense lamellar system comparable to that in control low light plastids. Thus photosynthetic parameters and chloroplast ultrastructure keep pace with or can be readjusted by prevailing light conditions even in fully differentiated leaves.

Limiting Factors in Photosynthesis- from the Chloroplast to the Plant Canopy

The efficiency for solar energy conversion in the most efficient crop like sugar-cane approaches 2 % when expressed on a yearly basis (Varlet-Granchet et al 1978). This yield could seem rather low when compared to the efficiency of the photochemical apparatus which is higher than 30 % under optimal conditions. Despite their low efficiency, plants are still interesting energy converters because they have solved a major problem for solar energy conversion which is the storage of energy under an easily usable form for various purposes (Calvin 1980, this volume). The aim of this paper is to review the main causes of decrease in yield from the chloroplast to the canopy and to look at the possible improvements. This analysis is limited to plants grown under non-limiting conditions for water, temperature and mineral nutrition. The effect of water stress and mineral deficiency is very important in natural ecosystems and is discussed more specifically by Tailing (1980, this volume) for fresh water population and by Divigneaud (1980, this volume) for terrestrial biocenosis.

Relationships Between Source Leaf Photosynthesis, Export and Grain Filling in Maize

Grain filling in Maize is mainly dependent on photosynthetic carbon fixed after pollination(l). However in spite of the high sink demand, photosynthetic rate tends to decline because of leaf senescence(2). This phenomenon is probably linked to N remobilization needed for grain protein synthesis.