Giacomo Novi

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.

HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana.

Plants often experience challenging hypoxic conditions imposed by soil waterlogging or complete flooding. In rice, Sub1A, a flooding-induced ethylene responsive factor (ERF) plays a crucial role in submergence tolerance. In this study, we examined two Arabidopsis Hypoxia Responsive ERF genes (HRE1 and HRE2), belonging to the same ERF group as Sub1A. Transgenic Arabidopsis plants, which over-expressed HRE1, showed an improved tolerance of anoxia, whereas a double-knockout mutant hre1hre2 was more susceptible than the wild type. HRE1 over-expressing plants showed an increased activity in the fermentative enzymes pyruvate decarboxylase and alcohol dehydrogenase together with increased ethanol production under hypoxia, but not in normoxia. Whole-genome microarray analyses suggested that an over-expression of HRE1, but not HRE2, increased the induction of most anaerobic genes under hypoxia. Real-time quantitative (q)PCR analyses confirmed a positive effect of HRE1 over-expression on several anaerobic genes, whereas the double-knockout mutant hre1hre2 showed a decreased expression in the same genes after 4 h of hypoxia. Single-knockout mutants did not show significant differences from the wild type. We found that the regulation of HRE1 and HRE2 by low oxygen relies on different mechanisms, since HRE1 requires protein synthesis to be induced while HRE2 does not. HRE2 is likely to be regulated post-transcriptionally by mRNA stabilization. We propose that HRE1 and HRE2 play a partially redundant role in low oxygen signalling in Arabidopsis thaliana, thus improving the tolerance of the plant to the stress by enhancing anaerobic gene expression and ethanolic fermentation.

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.

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.

Reactive Oxygen Species-Driven Transcription in Arabidopsis under Oxygen Deprivation

Reactive oxygen species (ROS) play an important role as triggers of gene expression during biotic and abiotic stresses, among which is low oxygen (O2). Previous studies have shown that ROS regulation under low O2 is driven by a RHO-like GTPase that allows tight control of hydrogen peroxide (H2O2) production. H2O2 is thought to regulate the expression of heat shock proteins, in a mechanism that is common to both O2 deprivation and to heat stress. In this work, we used publicly available Arabidopsis (Arabidopsis thaliana) microarray datasets related to ROS and O2 deprivation to define transcriptome convergence pattern. Our results show that although Arabidopsis response to anoxic and hypoxic treatments share a common core of genes related to the anaerobic metabolism, they differ in terms of ROS-related gene response. We propose that H2O2 production under O2 deprivation is a trait present in a very early phase of anoxia, and that ROS are needed for the regulation of a set of genes belonging to the heat shock protein and ROS-mediated groups. This mechanism, likely not regulated via the N-end rule pathway for O2 sensing, is probably mediated by a NADPH oxidase and it is involved in plant tolerance to the stress.

Heat acclimation and cross‐tolerance against anoxia in Arabidopsis

Arabidopsis seedlings are highly sensitive to low oxygen and they die rapidly when exposed to anoxia. Tolerance to anoxia depends on the ability to efficiently use carbohydrates through the fermentative pathway, as highlighted by the lower tolerance displayed by a mutant devoid of alcohol dehydrogenase. Other mechanisms of tolerance are also possible and may include a role for heat-induced genes. In fact, heat shock proteins (HSPs) are induced by anoxia. This suggests that there may be a cross-adaptation mechanism between heat and anoxic stress, and in this work, we studied the acclimation of Arabidopsis seedlings both to low oxygen and heat. The results show that seedlings subjected to hypoxia or heat pretreatment survive anoxia much better. Interestingly, we also observed an increased anoxia tolerance in heat-treated alcohol dehydrogenase (adh) mutant plants. On the other hand, anoxic pretreatment does not confer tolerance to heat stress. The success of the induction of HSPs by anoxia is in direct relation to the amount of sucrose available, and this in turn relates to how well seedlings will survive under anoxia. HSP transcripts were also detected during seed development and germination, two hypoxia-prone processes, suggesting that hypoxia-induced HSP expression is physiologically relevant.

SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood-tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A-genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance-related SUB1A-1 and the intolerance-related SUB1A-2 alleles were found in tolerant and sensitive accessions, respectively. Flooding-tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C-genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence-induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A-1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C-genome group, which show instead a SUB1A-independent response to submergence.

Nighttime Sugar Starvation Orchestrates Gibberellin Biosynthesis and Plant Growth in Arabidopsis

This work demonstrates that plants integrate the efficiency of photosynthesis over a period of days and transduce that information into a daily rate of gibberellin synthesis. This enables a plant to match its growth rate to its environment without unnecessary short-term fluctuations. A plant’s eventual size depends on the integration of its genetic program with environmental cues, which vary on a daily basis. Both efficient carbon metabolism and the plant hormone gibberellin are required to guarantee optimal plant growth. Yet, little is known about the interplay between carbon metabolism and gibberellins that modulates plant growth. Here, we show that sugar starvation in Arabidopsis thaliana arising from inefficient starch metabolism at night strongly reduces the expression of ent-kaurene synthase, a key regulatory enzyme for gibberellin synthesis, the following day. Our results demonstrate that plants integrate the efficiency of photosynthesis over a period of days, which is transduced into a daily rate of gibberellin biosynthesis. This enables a plant to grow to a size that is compatible with its environment.

Low oxygen response mechanisms in green organisms

Low oxygen stress often occurs during the life of green organisms, mostly due to the environmental conditions affecting oxygen availability. Both plants and algae respond to low oxygen by resetting their metabolism. The shift from mitochondrial respiration to fermentation is the hallmark of anaerobic metabolism in most organisms. This involves a modified carbohydrate metabolism coupled with glycolysis and fermentation. For a coordinated response to low oxygen, plants exploit various molecular mechanisms to sense when oxygen is either absent or in limited amounts. In Arabidopsis thaliana, a direct oxygen sensing system has recently been discovered, where a conserved N-terminal motif on some ethylene responsive factors (ERFs), targets the fate of the protein under normoxia/hypoxia. In Oryza sativa, this same group of ERFs drives physiological and anatomical modifications that vary in relation to the genotype studied. The microalga Chlamydomonas reinhardtii responses to low oxygen seem to have evolved independently of higher plants, posing questions on how the fermentative metabolism is modulated. In this review, we summarize the most recent findings related to these topics, highlighting promising developments for the future.