Iron deficiency and the structure and physiology of maize chloroplasts

The ultrastructure of mesophyll chloroplasts of maize (Zea mays L.) was more severely affected by iron deficiency that induced mild chlorosis than was the ultrastructure of bundle sheath plastids. Ferredoxin and ribulose diphosphate carboxylase levels were severely decreased by iron deficiency. Malic enzyme was less affected, and phosphoenolpyruvate carboxylase activity remained high even under severe iron deficiency. Iron deficient leaves fixed carbon into malic and aspartic acids but the rate of entrance of carbon into the sugar phosphates and sucrose was greatly reduced compared to the control. Chlorophyll a/b ratios ranged from low values of less than 2 in severely iron deficient leaves to high values exceeding 4 in leaves showing little iron deficiency.     

Characterization and expression profile analysis of a sucrose synthase gene from common bean (Phaseolus vulgaris L.) during seed development

A full-length cDNA encoding common bean (Phaseolus vulgaris L.) sucrose synthase (designated as Pv_BAT93 Sus), which catalyses the synthesis and cleavage of sucrose, was isolated from seeds at 15 days after pollination (DAP) by rapid amplification of cDNA ends (RACE). The full-length cDNA of Pv_BAT93 Sus had a 2,418 bp open reading frame (ORF) encoding a protein of 806 amino acid residues. Sequence comparison analysis showed that Pv_BAT93 Sus was very similar to several members of the sucrose synthase family of other plant species. Tissue expression pattern analysis showed that Pv_BAT93 Sus was expressed in leaves, flowers, stems, roots, cotyledons, and particularly during seed development. Expression studies using in situ hybridization revealed altered spatial and temporal patterns of Sus expression in the EMS mutant relative to wild-type and confirmed Sus expression in common bean developing seeds. The expression and accumulation of Sus mRNA was clearly shown in several tissues, such as the suspensor and embryo, but also in the transfer cells and endothelium. The results highlight the diverse roles that Sus might play during seed development in common bean.     

Cloning, characterization and expression analysis of a sucrose synthase gene from tropical epiphytic orchid Oncidium Goldiana

A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic orchid Oncidium Goldiana. The cDNA is 2829 bp in length containing an open reading frame of 2447 bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of O . Goldiana sucrose synthase ( Osus ) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli . The Osus mRNA is present in all the tissues analysed, with the highest levels in strong sinks such as developing inflorescence and root tips. Incubation with sucrose or glucose resulted in a significant increase in the steady-state Osus mRNA levels in root tips and mature leaves in a similar pattern to maize Sus1 . Expression of the Osus mRNA in mature leaves was markedly enhanced by anaerobic conditions and elevated CO₂. The expression pattern and regulation of the gene suggest that the sucrose synthase plays an important role in the growth and development of the tropical epiphytic orchid O . Goldiana.     

Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza

Arbuscular mycorrhizal fungi enhance CO₂ assimilation of their hosts which ensure the demand for carbohydrates of these obligate biotrophic microorganisms. Photosynthetic parameters were measured in tomato colonised or not by the arbuscular mycorrhizal fungus Glomus mosseae. In addition, carbohydrate contents and mRNA accumulation of three sucrose transporter genes were analysed. Mycorrhizal plants showed increased opening of stomata and assimilated significant more CO₂. A higher proportion of the absorbed light was used for photochemical processes, while non-photochemical quenching and the content of photoprotective pigments were lower. Analysis of sugar contents showed no significant differences in leaves but enhanced levels of sucrose and fructose in roots, while glucose amounts stayed constant. The three sucrose transporter encoding genes of tomato SlSUT1, SlSUT2 and SlSUT4 were up-regulated providing transport capacities to transfer sucrose into the roots. It is proposed that a significant proportion of sugars is used by the mycorrhizal fungus, because only amounts of fructose were increased, while levels of glucose, which is mainly transferred towards the fungus, were nearly constant.     

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.     

Characterization of the Xanthophyll Cycle and Other Photosynthetic Pigment Changes Induced by Iron Deficiency in Sugar Beet (Beta vulgaris L.)

In this work we characterize the changes induced by iron deficiency in the pigment composition of sugar beet (Beta vulgaris L.) leaves. When sugar beet plants were grown hydroponically under limited iron supply, neoxanthin and β-carotene decreased concomitantly with chlorophyll a, whereas lutein and the carotenoids within the xanthophyll cycle were less affected. Iron deficiency caused major increases in the lutein/chlorophyll a and xanthophyll cycle pigments/chlorophyll a molar ratios. Xanthophyll cycle carotenoids in Fe-deficient plants underwent epoxidations and de-epoxidations in response to ambient light conditions. In dark adapted Fe-deficient plants most of the xanthophyll cycle pigment pool was in the epoxidated form violaxanthin. We show, both by HPLC and by in vivo 505 nanometers absorbance changes, that in Fe deficient plants and in response to light, the de-epoxidated forms antheraxanthin and zeaxanthin were rapidly formed at the expense of violaxanthin. Several hours after returning to dark, the xanthophyll cycle was shifted again toward violaxanthin. The ratio of variable to maximum chlorophyll fluorescence from intact leaves was decreased by iron deficiency. However, in iron deficient leaves this ratio was little affected by light conditions which displace the xanthophyll cycle toward epoxidation or de-epoxidation. This suggests that the functioning of the xanthophyll cycle is not necessarily linked to protection against excess light input.     

A Phosphate Transporter Promoter from Arabidopsis thaliana AtPHT1;4 Gene Drives Preferential Gene Expression in Transgenic Maize Roots Under Phosphorus Starvation

Phosphorus (P) stress responsive genes have been identified and characterized, including the high-affinity phosphate transporter AtPHT1;4 from Arabidopsis thaliana. This gene encodes a membrane protein that is primarily expressed in roots under phosphorus deficiency. A 2.3-kb promoter region from AtPHT1;4 has been fused with the β-glucuronidase (GUS) encoding gene and introduced into maize via biolistic bombardment to evaluate its spatiotemporal activity in a heterologous system. AtPHT1;4::GUS expression is detected preferentially in transgenic maize roots under P deficiency. Further analysis of transgenic plants has also revealed that GUS activity is higher in roots than in leaves by about sixfold. These results demonstrate the ability of AtPHT1;4 promoter to direct expression of the reporter gene in a monocot root system under P stress. This property of AtPHT1;4 promoter makes it useful to engineer maize plants to modify the soil’s rhizosphere and increase efficiency of P acquisition under P stress conditions.     

Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis

In maize, two paralogous genes, Sh1 and Sus1, encode two biochemically similar isozymes of sucrose synthase, SS1 and SS2, respectively. Previous studies have attributed the mild starch deficiency of the shrunken1 (sh1) endosperm to the loss of the SS1 isozyme in the mutant. Here we describe the first mutation in the sucrose synthase1 (Sus1) gene, sus1-1, and the isolation of a double recessive genotype, sh1 sus1-1. Combined data from diverse studies, including Northern and Western analyses, RT-PCR and genomic PCR, cloning and sequencing data for the 3′ region, show that the mutant sus1-1 gene has a complex pattern of expression, albeit at much reduced levels as compared to the Sus1 gene. Endosperm sucrose synthase activity in sh1 sus1-1 was barely 0.5% of the total activity in the Sh1 Sus1 genotype. Significantly, comparative analyses of Sh1 Sus1, sh1 Sus1 and sh1 sus1-1 genotypes have, for the first time, allowed us to dissect the relative contributions of each isozyme to endosperm development. Starch contents in endosperm of the three related genotypes were 100, 78 and 53%, respectively. Anatomical analyses, which confirmed the previously described early cell degeneration phenotype unique to the sh1 Sus1 endosperm, revealed no detectable difference between the two sh1 genotypes. We conclude that the SS1 isozyme plays the dominant role in providing the substrate for cellulose biosynthesis, whereas the SS2 protein is needed mainly for generating precursors for starch biosynthesis.     

Sulla carnosa modulates root invertase activity in response to the inhibition of long‐distance sucrose transport under magnesium deficiency

• Being the principal product of photosynthesis, sucrose is involved in many metabolic processes in plants. As magnesium (Mg) is phloem mobile, an inverse relationship between Mg shortage and sugar accumulation in leaves is often observed.
• Mg deficiency effects on carbohydrate contents and invertase activities were determined in Sulla carnosa Desf. Plants were grown hydroponically at different Mg concentrations (0.00, 0.01, 0.05 and 1.50 mM Mg) for one month.
• Mineral analysis showed that Mg contents were drastically diminished in shoots and roots mainly at 0.01 and 0.00 mM Mg. This decline was adversely associated with a significant increase of sucrose, fructose and mainly glucose in shoots of plants exposed to severe deficiency. By contrast, sugar contents were severely reduced in roots of these plants indicating an alteration of carbohydrate partitioning between shoots and roots of Mg‐deficient plants. Cell wall invertase activity was highly enhanced in roots of Mg‐deficient plants, while the vacuolar invertase activity was reduced at 0.00 mM Mg. This decrease of vacuolar invertase activity may indicate the sensibility of roots to Mg starvation resulting from sucrose transport inhibition. ¹⁴CO₂ labeling experiments were in accordance with these findings showing an inhibition of sucrose transport from source leaves to sink tissues (roots) under Mg depletion.
• The obtained results confirm previous findings about Mg involvement in photosynthate loading into phloem and add new insights into mechanisms evolved by S. carnosa to cope with Mg shortage in particular the increase of the activity of cell wall invertase.

     

Magnesium deficiency in sugar beets alters sugar partitioning and phloem loading in young mature leaves

Magnesium deficiency has been reported to affect plant growth and biomass partitioning between root and shoot. The present work aims to identify how Mg deficiency alters carbon partitioning in sugar beet (Beta vulgaris L.) plants. Fresh biomass, Mg and sugar contents were followed in diverse organs over 20 days under Mg-sufficient and Mg-deficient conditions. At the end of the treatment, the aerial biomass, but not the root biomass, of Mg-deficient plants was lower compared to control plants. A clear inverse relationship between Mg and sugar contents in leaves was found. Mg deficiency promoted a marked increase in sucrose and starch accumulation in the uppermost expanded leaves, which also had the lowest content of Mg among all the leaves of the rosette. The oldest leaves maintained a higher Mg content. [¹⁴C]Sucrose labelling showed that sucrose export from the uppermost expanded leaves was inhibited. In contrast, sucrose export from the oldest leaves, which are close to, and export mainly to, the roots, was not restricted. In response to Mg deficiency, the BvSUT1 gene encoding a companion cell sucrose/H⁺ symporter was induced in the uppermost expanded leaves, but without further enhancement of sucrose loading into the phloem. The observed increase in BvSUT1 gene expression supports the idea that sucrose loading into the phloem is defective, resulting in its accumulation in the leaf.     

Partitioning of Sugar between Growth and Nitrate Reduction in Cotton Roots

The level of endogenous sugars was inversely related to nitrate availability in young cotton (Gossypium hirsutum L.) plants, with high nitrate causing a greater decline in sugar content of roots than of shoots. High nitrate (low sugar) plants also displayed relatively more shoot growth and less root growth than low nitrate (high sugar) plants. These data are consistent with the theory that roots are poor competitors for sugar, and that sugar supply is a major factor limiting root growth in vivo.

The effects of endogenous sugar level on root growth and on nitrate reductase activity in the root were different. When root sugar level was experimentally controlled by varying nitrate concentration in the nutrient solution, root growth was less sensitive than nitrate reductase activity to sugar deficiency. Also, in sterile root tips cultured on media containing a wide range of sucrose concentrations, growth rate was considerably less sensitive to endogenous sugar deficiency than was nitrate assimilation rate. Similarly, in plants which were detopped or girdled, nitrate reductase activity in the roots declined more rapidly than did root sugars, especially glucose and fructose. These results suggest that when sugar is deficient, cotton roots preferentially use it for growth at the expense of nitrate reduction.

     

Substrate inhibition of maize endosperm sucrose synthase by fructose and its interaction with glucose inhibition

Sucrose synthase (EC 2.4.1.13) was purified to homogeneity from developing maize (Zea mays L.) endosperm. Substrate saturation and inhibitor kinetics were examined for the sucrose synthase reaction. The K_m-values for fructose and uridine diphosphate glucose (UDPGlc) were estimated to be 7.8 mM and 76 μM, respectively. Fructose concentrations over 20 mM inhibited sucrose synthase in an uncompetitive manner with respect to UDPGlc. Glucose was also found to be an uncompetitive inhibitor with respect to both fructose and UDPGlc. At inhibitory concentrations of fructose, the apparent K_i for glucose increased linearly with increasing fructose concentration. The results suggest an ordered kinetic mechanism for sucrose synthase where UDPGlc binds first and UDP dissociates last. Fructose and glucose both inhibit by binding to the enzyme-UDP complex. Fructose and glucose, which are present in maize endosperm as the products of invertase, could inhibit sucrose synthase, especially in basal regions of the kernel where hexosesmay accumulate.     

Antisense repression of sucrose synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning

To unravel the roles of sucrose synthase in carrot, we reduced its activity in transgenic carrot plants by an antisense approach. For this purpose, the cDNA for the main form of carrot sucrose synthase was expressed in antisense orientation behind the 35S promoter of cauliflower mosaic virus. In independent antisense plant lines grown in soil, sucrose synthase activity was reduced in tap roots but not in leaves. In the sink organs, sucrose utilization was markedly decreased and higher levels of sucrose but lower levels of UDP-glucose, glucose, fructose, starch and cellulose were found. The phenotype of the antisense plants clearly differed from that of control plants. Both leaves and roots were markedly smaller, and the antisense line with the lowest sucrose synthase activity also developed the smallest plants. In most of the plant lines, the leaf-to-root dry weight ratios were not changed, suggesting that sucrose synthase in carrot is a major determinant of plant growth rather than of sucrose partitioning. In contrast to the acid invertases, which are critical for partitioning of assimilated carbon between source leaves and tap roots (Tang et al., Plant Cell 11: 177–189 (1999)), sucrose synthase appears to be the main sucrose-cleaving activity, feeding sucrose into metabolism.