Rice domestication did more than turn a wild grass into a crop. Over generations of cultivation and selection, rice developed traits that made it more useful to humans, including changes in grain size, yield, flowering time and seed shattering. But how these changes emerged at the molecular level is still not completely understood.
A recent study at NCBS has identified a regulatory system involving copper, small RNA molecules and a transcription factor that contribute to the domestication of indica rice.
The researchers compared the wild rice Oryza nivara with the cultivated indica rice variety Pusa Basmati 1 (PB-1). They found major differences in genes involved in the response to nutrients and metals, with copper-associated genes forming one of the prominent groups of genes that differed between the two rice types. Around 25% of the rice genome (quantitative trait loci) analysed overlapped with copper-responsive genes. This points to a possible connection between copper regulation and domestication-associated traits.
"Copper is needed by plants in trace amounts, as it is a micronutrient. The role of micronutrients in crop domestication is unexplored and our study addresses this gap" says Steffi Raju, the lead author of the study. "Copper is used by several proteins involved in processes such as metabolism and photosynthesis. But too little or too much copper can be harmful, so plants need to carefully control how much copper they take up, move around and store," she added.
One way plants do this is through microRNAs (miRNAs). These are short RNA molecules that regulate gene activity by targeting specific messenger RNAs. In rice, four
copper-responsive miRNAs (miR397, miR398, miR408 and miR528) were found to behave differently between wild and cultivated rice.
These miRNAs were more abundant in Oryza nivara. Their targets include genes coding for laccases, uclacyanins, multicopper oxidases and copper/zinc superoxide dismutases. Many of which are involved in copper-related processes. Steffi and team found that the levels of these target genes tended to decrease as their corresponding miRNAs spike, indicating their role in regulating these genes.
Some of these targets have already been linked to traits important for rice yield. For example, miR397 regulates laccases, while miR408 and miR398 regulate uclacyanins and Cu/Zn-SODs respectively.
The research team then looked for the switch controlling this network
They focused on OsSPL9, a transcription factor. OsSPL9 belongs to the SPL family of transcription factors and is the rice counterpart of a copper-responsive regulator called SPL7 in the plant Arabidopsis.
When the team increased the amount of OsSPL9 in cultivated rice, all four copper-associated miRNAs increased by at least two fold. Knocking out OsSPL9 caused these miRNAs to become undetectable. This established OsSPL9 as an upstream regulator of the four miRNAs.
Changing OsSPL9 also changed the rice plant itself. Plants with too much OsSPL9 developed more tillers but had smaller panicles, abnormal grain filling and smaller seeds. Plants in which OsSPL9 was knocked out also showed reduced panicle size and smaller, abnormal seeds. The results suggest that rice needs the right amount of OsSPL9 for normal growth and yield.
Steffi and team next asked how OsSPL9 controls these genes. Using ChIP-seq, they found that OsSPL9 binds to thousands of regions in the rice genome. Many of these regions contain a short DNA sequence called a GTAC motif. OsSPL9 was found near several copper-associated genes and miRNA genes, and experiments confirmed that the transcription factor can directly bind to GTAC-containing DNA sequences.
“The relationship did not stop with OsSPL9 controlling the miRNAs. We found evidence that the miRNAs can influence the same copper regulatory system in return. When individual copper associated miRNAs such as miR397, miR528 and miR408 were knocked out, the plants developed severe growth defects. Their panicles were shorter, seed filling was reduced and seeds were smaller. Some of these mutants also showed reduced copper accumulation, similar to plants in which OsSPL9 had been disrupted,” says Steffi.
“Knocking out one miRNA could also reduce the levels of other copper associated miRNAs,” she added. This suggests that these molecules do not function as isolated switches. Instead, they appear to form an interconnected feedback system involving OsSPL9, copper availability and the genes targeted by the miRNAs.
The researchers also found differences in copper accumulation itself. Oryza nivara accumulated more copper than PB-1, while several copper transporters were more highly expressed in cultivated rice. When SPL9 levels are increased in cultivated rice, the copper accumulation increases as observed similar to wild rice. These results place copper availability at the centre of the regulatory network connecting OsSPL9, miRNAs and plant traits.
“How nutrient availability and habitat altered molecular wiring in rice during domestication was previously unknown. Several epigenetic changes characterised in our study are unique and help to improve rice as a high-yielding crop,” says Prof PV Shivaprasad, the Principal Investigator of the study.
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