In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.

While plant rRNA gene organization and expression have been studied for several decades, the repetitive nature and high sequence identity between the tandemly-repeated units has prevented precise studies to determine which units are active and elucidate mechanisms of spatial and temporal transcriptional control. We have detected four variants among rRNA genes of Arabidopsis thaliana by analysis of the 3' external spacer region. Surprisingly, the most abundant variant, representing ~50% of the genes, is not expressed in wild-type plants but is transcribed in lines mutated in one of the two nucleolin genes.1 Analysis of Arabidopsis ecotypes indicated considerable variability in numbers and presence/absence of variants, although more closely-related ecotypes show similar profiles. Sequence analysis showed that one of the variants is not only unexpectedly located within a 5S RNA gene cluster in the pericentromeric region of chromosome 3 but is also potentially highly expressed in wild-type plants. We present a model to explain how transcription of this this unusual variant, maintained in an active state by nucleolin binding, could be involved in control of expression of the major variant, while absence of nucleolin leads to silencing of the minor variant and consequent expression of the major variant.

Humic and oxalic acids have the effects of promoting plant growth. We test whether they are able to positively impact wheat yield under newly reclaimed sandy soil, where water deficiency negatively influences yield. Foliar application of humic acid and oxalic acid on two wheat cultivars, Gemiza-9 and Sakha-93, leads to overall better performance of the plants and increases the yield significantly, irrespective of the cultivar genetic background. However, Gemiza-9 surpassed Sakha-93 in grain yield parameters. The highest values of grain and protein yields/ha were obtained in both cultivars, when the plants were sprayed with a combination of 17 mg/L humic acid and (300 mg/L) oxalic acid. Humic and oxalic acid showed accumulative yield-promoting effect. To understand the mechanism by which humic and oxalic acids promoted grain yield, we performed SDS-PAGE followed by MS-MS-LC analyses. We identified a unique humic acid-induced 52 KDa band in Gemiza-9. The band contained three major proteins, Ribulose bisphosphate carboxylase large chain, ADP-glucose synthase and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN). Thus humic acid increased the activity of plastid enzymes involved in photosynthesis, sucrose biosynthesis and starched accumulation to improve the overall performance of the plant.

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