The Qin Heji research group and collaborators of the College of Life Sciences have discovered important new factors regulating the formation of plant thermal morphology

The past summer of 2022 can be said to be the hottest summer in the history of meteorological observation, and the high temperature in many parts of China has reached unprecedented historical extreme levels
.
Not only China, but also many countries in the world have experienced record-breaking extreme high temperature weather, and even in the Arctic Circle, there has been a high temperature
of more than 32 ° C.
The increase in the frequency and duration of extreme weather has made global warming a common problem faced by all countries, seriously affecting crop production and world food security
.
The molecular mechanism of plant response to high temperatures has become an important scientific question
of great concern to botanists.
Plants that cannot move have evolved strategies to adapt to
habitats by malleating them according to the surrounding environmental conditions.
At high temperatures, the hypocotyl and petiole elongation of plants, the angle between the leaf growth direction and the ground increases, and phenotypes such as early flowers appear, which are collectively referred to as the thermal form of plants
.
The construction of the hot form is conducive to the plant to reduce its own temperature and better adapt to the high temperature environment
.
Scientists have found that the bHLH transcription factor PIF4 plays a key regulatory role in the construction of plant thermal morphology, and PIF4 itself is strictly regulated by multiple factors, such as the transcription factor TCP5 can enhance the transcriptional activation of PIF4 transcription factor
.
However, the mechanism of fine regulation of PIF4 transcription factors is still not well understood
.

On September 24, 2022, Professor Qin Heji's research group and collaborators from the School of Life Sciences of Peking University published a research paper entitled "Activation Tagging Identifies WRKY14 as a Repressor of Plant Thermomorphogenesis in Arabidopsis" online in the internationally renowned academic journal Molecular Plant.
It was found that ABT1/WRKY14 interacted with transcription factor TCP5 to finely regulate the activity of PIF4 transcription factor, thereby controlling the thermal morphology of plants, and revealed that WRKY14, TCP5 and PIF4 form complex regulatory modules to fine-tune the activity of PIF4 to make plants respond to different temperature changes
.

When Qin Heji's research group studied the function of the TCP transcription factor family, it was found that TCP5 regulates plant thermal morphology by promoting PIF4 transcription factor function at the transcription level and protein level
.
In order to find new components that regulate the formation of plant thermal morphology, mutant abt1-D
was obtained by screening Arabidopsis T-DNA insertion activation mutant library according to the phenotype of plant hypocotyl lengthening at high temperature.
Compared to wild-type controls, the hypocotyl of abt1-D is not elongated at high temperatures (Figures 1A-1E).

They found through T-DNA insertion location analysis that T-DNA was inserted into the upstream promoter region of the ABT1 gene in this mutant and co-separated from the phenotype of ABT1-D (Figures 1F and 1G).

Since the T-DNA has four enhancers of 35S promoters, the expression level of the downstream gene ABT1 at the insertion site is significantly increased (Figure 1H), and it was proved that ABT1 plays an important role in regulating the construction of plant thermal morphology by reproducing the phenotypic experiment of overexpressing ABT1 with 35S promoter (Figure 1I-1L).

Figure 1.
T-DNA insertion activates mutant screening and found that ABT1 gene plays a key role
in regulating plant thermal morphology.
(A-E)T-DNA INSERTION ACTIVATES THE MUTANT ABT1-D WITH A PHENOTYPE THAT IS INSENSITIVE TO HIGH TEMPERATURES; (F) Schematic diagram of T-DNA insertion position in mutant abt1-D; (G) T-DNA insertion site co-separated from the phenotype of mutant abt1-D; (H) T-DNA insertion leads to overexpression of ABT1 gene; (I-L) The ABT1 driver with a 35S promoter can reproduce the otype of abt1-D that is not sensitive to high temperatures

Through bioinformatics analysis, the researchers found that ABT1 encodes WRKY14, which belongs to the WRKY transcription factor family
unique to plants.
The amino acid sequence of WRKY14 is highly similar to the amino acid sequence of WRKY35, WRKY65 and WRKY69 and belongs to the same clade in the phylogenetic tree, but the function of these WRKY is unknown
.
Transgenic lines overexpressing ABT2/WRKY35, ABT3/WRKY65 or ABT4/WRKY69 all exhibited a similar heat-insensitive phenotype to ABT1-D, indicating that the functions of the proteins encoded by these genes were similar
.
They determined that the genes had overlapping expression patterns by cloning the promoters of these genes and driving the GUS reporter genes, implying that the genes were likely functionally redundant
.
Indeed, by constructing the ABTmultiplex mutant, the research team found that the abt1abt2 abt3abt4 quadruple mutant exhibits the opposite phenotype of abt1-D, that is, more sensitive to high temperatures and has a longer
hypocotika at high temperatures.
The researchers further demonstrated that ABT1 can interact
with TCP5 through experiments such as yeast double hybridization, luciferase complementation and Co-IP.
Through a series of biochemical experiments, the researchers revealed the molecular mechanism by which ABT1 regulates the formation of plant thermal morphology, that is, the interaction between ABT1 and TCP5, which on the one hand inhibits the expression of PIF4 at the transcriptional level, and on the other hand, inhibits the formation and activity of PIF4-TCP5 transcription-activating complex by competing with PIF4 for TCP5 (Figure 2), thereby finely regulating the function of PIF4 transcription factors and plant response
to high temperature 。 Through a series of genetic interaction experiments, the researchers also proved that ABT1 does have the opposite effect with the positive regulators PIF4, TCP5 and BZR1 in the construction of thermal morphology, and is an important negative regulator in the construction of plant thermal morphology
.

Figure 2.
Working model of
the ABT1 gene.
High temperature inhibits the expression of ABT1, and ABT1 interacts with TCP5 to negatively regulate PIF4 transcription and PIF4-TCP5 transcription-activating complex formation and activity

This study not only discovered a new negative regulator WRKY14/ABT1 that regulates plant thermal morphology through positive genetic screening, but also revealed a fine regulatory mechanism
of a new PIF4 transcription factor.
Through the synergistic fine regulation between ABT1, TCP5 and PIF4, plants can respond to different temperatures and different high temperature durations to accurately regulate the thermal form of plants to adapt to changing ambient temperatures
.

Qin Wenqi, a doctoral student at South China Agricultural University, and Wang Ning, a doctoral student at Peking University, are the co-first authors
of the paper.
Professor Wu Aimin and Qin Heji of South China Agricultural University are the co-corresponding authors of the paper, and Yin Qi and Associate Professor Li Huiling, doctoral students of South China Agricultural University, also participated in the study
.
The research was supported
by the National Science Foundation for Outstanding Young Scholars (31725005), the National Natural Science Foundation of China (31970194), the National Key Research and Development Program of China (2017YFA0503800), and the Open Project of the State Key Laboratory of Protein and Plant Gene Research of Peking University.