Amedeo Alpi

Sucrose-Specific Induction of the Anthocyanin Biosynthetic Pathway in Arabidopsis

Sugars act as signaling molecules, whose signal transduction pathways may lead to the activation or inactivation of gene expression. Whole-genome transcript profiling reveals that the flavonoid and anthocyanin biosynthetic pathways are strongly up-regulated following sucrose (Suc) treatment. Besides mRNA accumulation, Suc affects both flavonoid and anthocyanin contents. We investigated the effects of sugars (Suc, glucose, and fructose) on genes coding for flavonoid and anthocyanin biosynthetic enzymes in Arabidopsis (Arabidopsis thaliana). The results indicate that the sugar-dependent up-regulation of the anthocyanin synthesis pathway is Suc specific. An altered induction of several anthocyanin biosynthetic genes, consistent with in vivo sugar modulation of mRNA accumulation, is observed in the phosphoglucomutase Arabidopsis mutant accumulating high levels of soluble sugars.

Plant responses to anaerobiosis

Abstract Plant life is greatly impaired under conditions of oxygen deficit. Only few plants can grow in waterlogged soils, thanks to anatomical adaptation allowing the transport of oxygen to the submerged parts of the plant. The behaviour of a few plant species able to germinate under completely anoxic conditions can be explained only by assuming that a biochemical adaptation is present in these species but absent in all the other species. Alternatively, plants that are tolerant to anoxia may avoid anoxia-induced biochemical changes that may be harmful to their life. In this paper we review the literature with the aim of presenting an updated status on some aspects of research on the responses opposed by plants to anaerobic stress.

Transcript Profiling of the Anoxic Rice Coleoptile

Rice (Oryza sativa) seeds can germinate in the complete absence of oxygen. Under anoxia, the rice coleoptile elongates, reaching a length greater than that of the aerobic one. In this article, we compared and investigated the transcriptome of rice coleoptiles grown under aerobic and anaerobic conditions. The results allow drawing a detailed picture of the modulation of the transcripts involved in anaerobic carbohydrate metabolism, suggesting up-regulation of the steps required to produce and metabolize pyruvate and its derivatives. Sugars appear to play a signaling role under anoxia, with several genes indirectly up-regulated by anoxia-driven sugar starvation. Analysis of the effects of anoxia on the expansin gene families revealed that EXPA7 and EXPB12 are likely to be involved in rice coleoptile elongation under anoxia. Genes coding for ethylene response factors and heat shock proteins are among the genes modulated by anoxia in both rice and Arabidopsis (Arabidopsis thaliana). Identification of anoxia-induced ethylene response factors is suggestive because genes belonging to this gene family play a crucial role in rice tolerance to submergence, a process closely related to, but independent from, the ability to germinate under anoxia. Genes coding for some enzymes requiring oxygen for their activity are dramatically down-regulated under anoxia, suggesting the existence of an energy-saving strategy in the regulation of gene expression.

A Genome-Wide Analysis of the Effects of Sucrose on Gene Expression in Arabidopsis Seedlings under Anoxia

Exogenous sucrose (Suc) greatly enhances anoxia tolerance of Arabidopsis (Arabidopsis thaliana) seedlings. We used the Affymetrix ATH1 GeneChip containing more than 22,500 probe sets to explore the anaerobic transcriptome of Arabidopsis seedlings kept under anoxia for 6 h in presence or absence of exogenous Suc. Functional clustering was performed using the MapMan software. Besides the expected induction of genes encoding enzymes involved in Suc metabolism and alcoholic fermentation, a large number of genes not related to these pathways were affected by anoxia. Addition of exogenous Suc mitigated the effects of anoxia on auxin responsive genes that are repressed under oxygen deprivation. Anoxia-induced Suc synthases showed a lower induction in presence of exogenous Suc, suggesting that induction of these genes might be related to an anoxia-dependent sugar starvation. Anoxic induction of genes coding for heat shock proteins was much stronger in presence of exogenous Suc. Interestingly, a short heat treatment enhanced anoxia tolerance, suggesting that heat shock proteins may play a role in survival to low oxygen. These results provide insight into the effects of Suc on the anoxic transcriptome and provide a list of candidate genes that enhance anoxia tolerance of Suc-treated seedlings.

Gibberellins, jasmonate and abscisic acid modulate the sucrose‐induced expression of anthocyanin biosynthetic genes in Arabidopsis

Anthocyanins are secondary metabolites, which play an important role in the physiology of plants. Both sucrose and hormones regulate anthocyanin synthesis. Here, the interplay between sucrose and plant hormones was investigated in the expression of sucrose-regulated genes coding for anthocyanin biosynthetic enzymes in Arabidopsis seedlings. The expression pattern of 14 genes involved in the anthocyanin biosynthetic pathway, including two transcription factors (PAP1, PAP2), was analysed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in Arabidopsis seedlings treated with sucrose and plant hormones. Sucrose-induction of the anthocyanin synthesis pathway was repressed by the addition of gibberellic acid (GA) whereas jasmonate (JA) and abscisic acid (ABA) had a synergic effect with sucrose. The gai mutant was less sensitive to GA-dependent repression of dihydroflavonol reductase. This would seem to prove that GAI signalling is involved in the crosstalk between sucrose and GA in wild-type Arabidopsis seedlings. Conversely, the inductive effect of sucrose was not strictly ABA mediated. Sucrose induction of anthocyanin genes required the COI1 gene, but not JAR1, which suggests a possible convergence of the jasmonate- and sucrose-signalling pathways. The results suggest the existence of a crosstalk between the sucrose and hormone signalling pathways in the regulation of the anthocyanin biosynthetic pathway.

Effect of Anoxia on Carbohydrate Metabolism in Rice Seedlings.

The metabolism of carbohydrates was investigated in rice (Oryza sativa L.) seedlings grown under anoxia. Two phases can be recognized in the utilization of carbohydrates: during the first days of germination under anoxia, the metabolism of sugars is mainly degradative, whereas after the induction of [alpha]-amylase (EC 3.2.1.1) has taken place, the increased presence of glucose and sucrose indicates that both starch degradation and sucrose synthesis operate. The analysis of the enzymes involved in carbohydrate metabolism indicates that anoxic rice seedlings possess a set of enzymes that allow the efficient metabolism of starch and sucrose to fructose-6-phosphate. We propose that cytosolic sucrose metabolism in anoxic rice seedlings takes place mainly through a sucrose synthase (EC 2.4.1.13) pathway with nucleoside diphosphate kinase (EC 2.7.4.6), allowing the cycling of urydilates needed for the operation of this pathway.

Amylolytic Activities in Cereal Seeds under Aerobic and Anaerobic Conditions

An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound. Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of [alpha]-amylase (EC 3.2.1.1), [beta]-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and [alpha]-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely. We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia. Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo. This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis ([alpha]-amylase, [beta]-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, [alpha]-glucosidase). These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions.

The Heat-Inducible Transcription Factor HsfA2 Enhances Anoxia Tolerance in Arabidopsis

Anoxia induces several heat shock proteins, and a mild heat pretreatment can acclimatize Arabidopsis (Arabidopsis thaliana) seedlings to subsequent anoxic treatment. In this study, we analyzed the response of Arabidopsis seedlings to anoxia, heat, and combined heat + anoxia stress. A significant overlap between the anoxic and the heat responses was observed by whole-genome microarray analysis. Among the transcription factors induced by both heat and anoxia, the heat shock factor A2 (HsfA2), known to be involved in Arabidopsis acclimation to heat and to other abiotic stresses, was strongly induced by anoxia. Heat-dependent acclimation to anoxia is lost in an HsfA2 knockout mutant (hsfa2) as well as in a double mutant for the constitutively expressed HsfA1a/HsfA1b (hsfA1a/1b), indicating that these three heat shock factors cooperate to confer anoxia tolerance. Arabidopsis seedlings that overexpress HsfA2 showed an increased expression of several known targets of this transcription factor and were markedly more tolerant to anoxia as well as to submergence. Anoxia failed to induce HsfA2 target proteins in wild-type seedlings, while overexpression of HsfA2 resulted in the production of HsfA2 targets under anoxia, correlating well with the low anoxia tolerance experiments. These results indicate that there is a considerable overlap between the molecular mechanisms of heat and anoxia tolerance and that HsfA2 is a player in these mechanisms.

Hormonal interplay during adventitious root formation in flooded tomato plants.

Soil flooding, which results in a decline in the availability of oxygen to submerged organs, negatively affects the growth and productivity of most crops. Although tomato (Solanum lycopersicum) is known for its sensitivity to waterlogging, its ability to produce adventitious roots (ARs) increases plant survival when the level of oxygen is decreased in the root zone. Ethylene entrapment by water may represent the first warning signal to the plant indicating waterlogging. We found that treatment with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) and the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) resulted in a reduction of AR formation in waterlogged plants. We observed that ethylene, perceived by the Never Ripe receptor, stimulated auxin transport. In a process requiring the Diageotropica gene, auxin accumulation in the stem triggered additional ethylene synthesis, which further stimulated a flux of auxin towards to the flooded parts of the plant. Auxin accumulation in the base of the plant induces growth of pre-formed root initials. This response of tomato plants results in a new root system that is capable of replacing the original one when it has been damaged by submergence.

Identification of sugar-modulated genes and evidence for in vivo sugar sensing in Arabidopsis

Sugar status regulates mechanisms controlling growth and development of plants. We studied the effects of sucrose at a genome-wide level in dark-grown 4-day-old Arabidopsis thaliana seedlings, identifying 797 genes strongly responsive to sucrose. Starting from the microarray analysis data, four up-regulated (At5g41670, At1g20950, At1g61800, and At2g28900) and four down-regulated (DIN6, At4g37220, At1g28330, and At1g74670) genes were chosen for further characterisation and as sugar sensing markers for in vivo analysis. The sugar modulation pattern of all eight genes was confirmed by real time RT-PCR analysis, revealing different concentration thresholds for sugar modulation. Finally, sugar-regulation of gene expression was demonstrated in vivo by using the starchless pgm mutant, which is unable to produce transitory starch. Sucrose-inducible genes are upregulated in pgm leaves at the end of a light treatment, when soluble sugars levels are higher than in the wild type. Conversely, sucrose-repressible genes show a higher expression at the end of the dark period in the mutant, when the levels of sugars in the leaf are lower. The results obtained indicate that the transcriptional response to exogenous sucrose allows the identification of genes displaying a pattern of expression in leaves compatible with their sugar-modulation in vivo.