Ultraviolet-B (UV-B) radiation affects leaf growth in a wide range of species. In this work, we demonstrate that UV-B levels present in solar radiation inhibit maize (Zea mays) leaf growth without causing any other visible stress symptoms, including the accumulation of DNA damage. We conducted kinematic analyses of cell division and expansion to understand the impact of UV-B radiation on these cellular processes. Our results demonstrate that the decrease in leaf growth in UV-B-irradiated leaves is a consequence of a reduction in cell production and a shortened growth zone (GZ). To determine the molecular pathways involved in UV-B inhibition of leaf growth, we performed RNA sequencing on isolated GZ tissues of control and UV-B-exposed plants. Our results show a link between the observed leaf growth inhibition and the expression of specific cell cycle and developmental genes, including growth-regulating factors (GRFs) and transcripts for proteins participating in different hormone pathways. Interestingly, the decrease in the GZ size correlates with a decrease in the concentration of GA19, the immediate precursor of the active gibberellin, GA1, by UV-B in this zone, which is regulated, at least in part, by the expression of GRF1 and possibly other transcription factors of the GRF family.
BackgroundAlong the root axis of Arabidopsis thaliana, cells pass through different developmental stages. In the apical meristem repeated cycles of division increase the numbers of cells. Upon leaving the meristem, these cells pass the transition zone where they are physiologically and mechanically prepared to undergo subsequent rapid elongation. During the process of elongation epidermal cells increase their length by 300% in a couple of hours. When elongation ceases, the cells acquire their final size, shape and functions (in the differentiation zone). Ethylene administered as its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is capable of inhibiting elongation in a concentration-dependent way. Using a microarray analysis, genes and/or processes involved in this elongation arrest are identified.ResultsUsing a CATMA-microarray analysis performed on control and 3h ACC-treated roots, 240 differentially expressed genes were identified. Quantitative Real-Time RT-PCR analysis of the 10 most up and down regulated genes combined with literature search confirmed the accurateness of the analysis. This revealed that inhibition of cell elongation is, at least partly, caused by restricting the events that under normal growth conditions initiate elongation and by increasing the processes that normally stop cellular elongation at the end of the elongation/onset of differentiation zone.ConclusionsACC interferes with cell elongation in the Arabidopsis thaliana roots by inhibiting cells from entering the elongation process and by immediately stimulating the formation of cross-links in cell wall components, diminishing the remaining elongation capacity. From the analysis of the differentially expressed genes, it becomes clear that many genes identified in this response, are also involved in several other kind of stress responses. This suggests that many responses originate from individual elicitors, but that somewhere in the downstream signaling cascade, these are converged to a ’common pathway’. Furthermore, several potential keyplayers, such as transcription factors and auxin-responsive genes, were identified by the microarray analysis. They await further analysis to reveal their exact role in the control of cell elongation.
The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.
It is concluded that AtEXLA2 may function as a positive regulator of cell elongation in the dark-grown hypocotyl of arabidopsis by possible interference with cellulose metabolism, deposition or its organization.
In plants many developmental processes are regulated by auxin and its directional transport. PINOID (PID) kinase helps to regulate this transport by influencing polar recruitment of PIN efflux proteins on the cellular membranes. We investigated how altered auxin levels affect leaf growth in Arabidopsis thaliana. Arabidopsis mutants and transgenic plants with altered PID expression levels were used to study the effect on auxin distribution and leaf development. Single knockouts showed small pleiotropic growth defects. Contrastingly, several leaf phenotypes related to changes in auxin concentrations and transcriptional activity were observed in PID overexpression (PIDOE) lines. Unlike in the knockout lines, the leaves of PIDOE lines showed an elevation in total indole-3-acetic acid (IAA). Accordingly, enhanced DR5-visualized auxin responses were detected, especially along the leaf margins. Kinematic analysis revealed that ectopic expression of PID negatively affects cell proliferation and expansion rates, yielding reduced cell numbers and small-sized cells in the PIDOE leaves. We used PIDOE lines as a tool to study auxin dose effects on leaf development and demonstrate that auxin, above a certain threshold, has a negative affect on leaf growth. RNA sequencing further showed how subtle PIDOE-related changes in auxin levels lead to transcriptional reprogramming of cellular processes.
The synthesis and composition of cell walls is dynamically adapted in response to many developmental and environmental signals. In this respect, cell wall proteins involved in controlling cell elongation are critical for cell development. Transcriptome analysis identified a gene in Arabidopsis thaliana, which was named proline-rich protein-like, AtPRPL1, based on sequence similarities from a phylogenetic analysis. The most resemblance was found to AtPRP1 and AtPRP3 from Arabidopsis, which are known to be involved in root hair growth and development. In A. thaliana four proline-rich cell wall protein genes, playing a role in building up the cross-connections between cell wall components, can be distinguished. AtPRPL1 is a small gene that in promoter::GUS (β-glucuronidase) analysis has high expression in trichoblast cells and in the collet. Chemical or mutational interference with root hair formation inhibited this expression. Altered expression levels in knock-out or overexpression lines interfered with normal root hair growth and etiolated hypocotyl development, but Fourier transform-infrared (FT-IR) analysis did not identify consistent changes in cell wall composition of root hairs and hypocotyl. Co-localization analysis of the AtPRPL1-green fluorescent protein (GFP) fusion protein and different red fluorescent protein (RFP)-labelled markers confirmed the presence of AtPRPL1-GFP in small vesicles moving over the endoplasmic reticulum. Together, these data indicate that the AtPRPL1 protein is involved in the cell's elongation process. How exactly this is achieved remains unclear at present.
The mutation causing the Silverblue color type (pp) is one of the most used recessive mutations within American mink (Neovison vison) fur farming, since it is involved in some of the popular color types such as Violet and Saphire which originate from a combination of recessive mutations. In the present study, the genomic and mRNA sequences of the melanophilin (MLPH) gene were studied in Violet, Silverblue and wild-type (wt) mink animals. Although breeding schemes and previous literature indicates that the Violet (aammpp) phenotype is a triple recessive color type involving the same locus as the Silverblue (pp) color type, our findings indicate different genotypes at the MLPH locus. Upon comparison at genomic level, we identified two deletions of the entire intron 7 and of the 5' end of intron 8 in the sequence of the Silverblue MLPH gene. When investigating the mRNA, the Silverblue animals completely lack exon 8, which encodes 65 residues, of which 47 define the Myosin Va (MYO5A) binding domain. This may cause the incorrect anchoring of the MLPH protein to MYO5A in Silverblue animals, resulting in an improper pigmentation as seen in diluted phenotypes. Additionally, in the MLPH mRNA of wt, Violet and Silverblue phenotypes, part of intron 8 is retained resulting in a truncated MLPH protein, which is 359 residues long in wt and Violet and 284 residues long in Silverblue. Subsequently, our findings point out that the missing actin-binding domain, in neither of the 3 analyzed phenotypes affects the transport of melanosomes or the consequent final pigmentation. Moreover, the loss of the major part of the MYO5A domain in the Silverblue MLPH protein seems to be the responsible for the dilute phenotype. Based on our genomic DNA data, genetic tests for selecting Silverblue and Violet carrier animals can be performed in American mink.
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