Unraveling the Intricate Nexus of Molecular Mechanisms Governing Rice Root Development: OsMPK3/6 and Auxin-Cytokinin Interplay

<div><p>The root system is an imperative component of a plant, involved in water and nutrient acquisition from the soil. Any subtle change in the root system may lead to drastic changes in plant productivity. Both auxin and cytokinin are implicated in regulating various root developmental aspects. One of the major signaling cascades facilitating various hormonal and developmental allocations is the Mitogen Activated Protein Kinase (MAPK) cascade. Innumerable efforts have been made to unravel the complex nexus involved in rice root development. In spite of a plethora of studies, a comprehensive study aiming to decipher the plausible cross-talk of MAPK signaling module with auxin and cytokinin signaling components in rice is missing. In the present study, extensive phenomics analysis of different stages of rice roots; transcript profiling by qRT-PCR of entire gene family of MAPK, MAPKK and PIN genes; as well as protein level and activity of potential MAPKs was investigated using western and immuno kinase assays both on auxin and cytokinin treatment. The above study led to the identification of various novel rice root specific phenotypic traits by using GiA roots software framework. High expression profile of OsMPK3/6, OsMKK4/5 and OsPIN 1b/9 and their marked transcript level modulation in response to both auxin and cytokinin was observed. Finally, the protein levels and activity assay further substantiated our present findings. Thus, OsMPK3/6-OsMKK4/5 module is elucidated as the putative, key player in auxin-cytokinin interaction augmenting their role by differentially regulating the expression patterns of OsPIN 1b/9 in root development in rice.</p></div

Differential transcript accumulation of all the components of MAPKs, MAPKKs and PINs.

<p>Absolute transcript levels of MAPKs (<b>A</b>), MAPKKs (<b>B</b>) and PIN (<b>C</b>) gene family members analyzed by qRT-PCR. Data shown is the average of three triplicates normalized to actin, with error bars indicating standard deviation.</p

Novel phenotypic variability on auxin and cytokinin treatment revealed by GiA Roots Software framework.

<p>Annotated images by GiA roots software of 7 days, 14 days, 21 days and 28 days old rice seedling treated with 1, 5μM IAA and BAP in comparison with the normally grown seedlings.</p

Regulation of MAP kinase signaling cascade by microRNAs in <i>Oryza sativa</i>

<div><p>Mitogen activated protein kinase (MAPK) pathway is one of the most conserved signaling cascade in plants regulating a plethora of cellular processes including normal growth and development, abiotic and biotic stress responses. The perception of external cues triggers the phosphorylation of three tier MAPKKK-MAPKK-MAPK cascade which finally modifies a downstream substrate thereby regulating the cellular processes. Whereas, the transcription regulation by MAPKs, mediated through their substrates is well studied in plants, the transcription and post-transcriptional regulation of the MAPK genes are poorly understood. Previous studies from the animals systems suggested the miRNAs regulate the post-transcriptional regulation of MAPK transcripts. Here we attempt to unravel the post-transcriptional regulation of MAPKs by miRNAs in model crop plant <i>Oryza sativa</i>. Using <i>in silico</i> tools, we predict the miRNAs for 98 out of 99 MAPK transcripts. The predicted miRNAs were validated for the biological relevance of their function. The inverse correlation between relative transcript levels between the MAPKs and their predicted miRNAs validated the <i>in silico</i> prediction. Taken together, this report demonstrates the significance of miRNAs in regulation of the MAPK pathway in plants with a new direction to study the plant signaling molecules.</p></div

Protein levels of OsMPK3 and OsMPK6 co-relate with the respective transcript profile.

<p>Western blots showing the protein levels of OsMPK3 (<b>A</b>) and OsMPK6 (<b>B</b>) in 7, 14, 21 and 28 days old rice roots upon IAA (1μM) and BAP (1μM) treatments. Anti-AtMPK3 and Anti-AtMPK6 antibodies were used to assess the respective protein levels of OsMPK3 and OsMPK6.</p

Phenotypic parameters show marked variability on auxin and cytokinin treatment.

<p>Graphical representation of root phenotypic parameters of untreated as well as 1, 5μM IAA and BAP treated 7, 14, 21 and 28 days rice seedlings. Average Root width (<b>A</b>), Network Depth (<b>B</b>), Maximum number of Roots (<b>C</b>), Specific Root Length (<b>D</b>) and Network Area (<b>E</b>) was analyzed using GiA roots software and plotted into bar graphs. Data shown is the average of three triplicates with error bars indicating standard deviation. The asterisks indicate a significant differences between Control vs. IAA treated and Control vs. BAP treated (***, P < 0.001), (*, P < 0.05) and (**, P < 0.01).</p

OsMPK3 and OsMPK6 show activation on auxin and cytokinin treatment in a contrasting manner.

<p>An immuno-kinase assay with pTEpY antibody, which specifically identifies the activated MAPK components, was performed. MAPK activity signals were observed at ~42, and ~44 kDa corresponding to the size of OsMPK3 and OsMPK6, respectively<b>.</b></p

Expression of MAPK, MAPKK and PIN gene family members show intricate regulation on auxin and cytokinin treatment.

<p>Transcripts level profile of OsMPK3 (<b>A</b>), OsMPK6 (<b>B</b>), OsMKK4 (<b>C</b>), OsMKK5 (<b>D</b>), OsPIN1b (<b>E</b>) OsPIN9 (<b>F</b>) and OsPIN2 (<b>G</b>) in response to auxin and cytokinin by qRT-PCR. Seedlings of 7, 14, 21 and 28 days were subjected to 1μM auxin and 1μM cytokinin treatments in ½ MS media. Expression level of untreated samples was taken as the baseline and all values shown are respective to baseline. Error bars indicate standard deviation of three independent experiments.</p

Verification of 3D structures of rice MAPKs and MAPKKs.

<p>DOPE–Discrete Optimized Protein Energy, PDF–Probability Density Function.</p

Ramachandran plot analysis of theoretical 3D structure of rice MAPKKs and MAPKs.

<p>The 3D Structures of eleven rice MAP kinases (OsMPK3, OsMPK4, OsMPK6, OsMPK7, OsMPK14, OsMPK16-1, OsMPK17-1, OsMPK20-2, OsMPK20-3, OsMPK20-5 and OsMPK21-2) and five MAP kinase kinases (OsMKK3, OsMKK4, OsMKK5, OsMKK6, OsMKK10-2) were validated using Ramachandran plot. The green dots/yellow dots show the amino acids that are in the most favoured regions and additionally allowed region while red dots show the amino acids that are in generously allowed region or disallowed regions. The regions covered by sky blue line show most favoured regions, while the regions covered by pink line show additionally allowed regions. Other regions of the plot show the generously allowed or disallowed region.</p