Discovering molecular 'team-work' underlying nitrate assimilation in a unicellular red alga

Date:
March 11, 2022
Source:
Tokyo Institute of Technology
Summary:
The mechanisms suppressing nitrate assimilation in plants under
nitrogen- repleted condition are poorly known, but researchers
may have made a breakthrough. They have recently studied the
transcription of nitrate assimilating genes in a unicellular red
alga and found that deletion of the negative domain of transcription
factor CmMYB1 or a previously unknown protein CmNDB1 is responsible
for alleviation of the transcription of nitrate assimilating genes
in nitrogen-repleted condition.



FULL STORY ========================================================================== Nitrogen is an essential component for plant growth and
development. Plants generally take up nitrogen from their environment
in the form of nitrates or ammonium and assimilate them into amino acids
with the help of the products of nitrate or ammonium assimilation genes, respectively. Transcription factors (TFs) regulate this activity, while
also modifying the rate of nitrate assimilation depending on the changes
in nitrogen levels. In nitrogen deficient condition, these TFs positively regulate the expression of nitrate assimilation genes.


==========================================================================
The same holds true for Cyanidioschyzon merolae,a unicellular red
alga, which serves as an excellent model of photosynthetic higher
organisms to study transcription. While it is known that the TF
called 'CmMYB1' is responsible for transcribing nitrate assimilation
genes in nitrogen-depleted condition, the mechanism for this under nitrogen-repleted(+N) condition is not clear.

To address this conundrum, in a study published in Frontiers in Plant
Science, a team of researchers from Tokyo Institute of Technology (Tokyo
Tech) and other institutes explored the molecular mechanisms controlling
the regulation of CmMYB1 in C. merolaeunder+N condition. "Based on our
previous studies, we were already aware that the activity of CmMYB1 is associated with a regulatory region of itself and/or CmMYB1-binding
protein(s), and thus attempted to identify it," explains Professor
Sousuke Imamura, the lead researcher of the study.

To do this, they first generated C. merolae strains with CmMYB1 knocked
out and transformed them with plasmids containing different truncated
sequences of CmMYB1. Subsequently, they identified a sequence of CmMYB1
between positions 311-380 as the key region responsible for downregulating nitrate assimilating genes under +N condition, inhibiting transcription
as a result. Further analyses based on quantitative polymerase chain
reactions indicated increased transcript levels of these genes in the
strain lacking the key region of CmMYB1 under +N condition. They labelled
this new sequence as the 'negative domain' (ND) of CmMYB1.

Moreover, they found that ND controls the subcellular localization
and promotor binding capacity of CmMYB1 under +N condition. Chromatin immunoprecipitation analyses confirmed the role of ND in reducing the
binding capacity of CmMYB1 to promoter regions of nitrate assimilating
genes under +N condition.

As the role of ND became clearer, the team decided to identify
proteins involved in its regulation. To do so, they constructed an
ND overexpressing strain, and performed immunoprecipitation and mass spectrometry analyses to obtain a list of potential ND binding proteins
in collaboration with investigators from Tohoku University. Finally,
through a yeast two-hybrid analysis, they identified a new protein with
an unknown function, named 'CmNDB1,' which interacts with ND of CmMYB1.

Through several analyses using a CmNDB1 knockout strain, they discovered
that CmNDB1 deletion resulted in nuclear localization of CmMYB1 and
reduced promoter binding capacity of CmMYB1 under +N condition. Moreover,
they discovered that CmNDB1 deletion increases the transcription of
nitrate assimilation genes under +N condition.

To sum up, both ND and CmNDB1 negatively control CmMYB1's activity
under +N condition, facilitate its localization in the cytoplasm,
and repress its binding to promoter regions of nitrate assimilating
genes to downregulate their transcription. Discussing the implications
of these findings, Professor Imamura says, "Ours is the first study
to reveal the mechanisms behind the regulation of transcription of
nitrate assimilation genes under +N condition. These innovative results
can significantly help in the advancement of research in this field." Identification of the critical ND region of CmMYB1 and the CmNDB1 protein
were key milestones that helped to understand the regulation of nitrate assimilation in C. merolae. Further studies, like investigation of the post-translational modifications of CmMYB1 and/or CmNDB1 are required
to better understand the regulation of nitrate assimilation not only in
this red alga but also in other photosynthetic organisms.


========================================================================== Story Source: Materials provided by Tokyo_Institute_of_Technology. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Baifeng Zhou, Hiroki Shima, Kazuhiko Igarashi, Kan Tanaka, Sousuke
Imamura. CmNDB1 and a Specific Domain of CmMYB1 Negatively Regulate
CmMYB1-Dependent Transcription of Nitrate Assimilation Genes Under
Nitrogen-Repleted Condition in a Unicellular Red Alga. Frontiers
in Plant Science, 2022; 13 DOI: 10.3389/fpls.2022.821947 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/03/220311095322.htm

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