Exploring the molecular hub in plant elongation responses: regulation of PHYs and PIFs

Light and temperature signalling response is a central mediator of plant growth plasticity. This thesis aimed to provides new molecular insights into control of plant growth. We investigated the role of phytochrome and PIF4 genes activity as function of light quality and temperature. First time the study shows that PHYD is a constitutive repressor of PHYA-reporter activity and that PHYB and PHYA reporter activity is strongly up-regulated under FR, while this response to FR is not affected by the classical FR sensor PHYA but by PHYB and PHYE. Our findings of the light sensitivity of phytochrome gene expression especially under the artificial LED light conditions provide crucial new fundamental insights that may be used to control plant growth, yield and quality in greenhouses and indoor farming industries. Next chapter, we discovered that the positive feedback regulation of PIF4 expression by BZR1 is actually broken by PIF4 itself, which acts as a negative regulator of its own expression. The negative action of PIF4 can also compete with the positive action of BZR1. Overall this adds a new layer to the regulation of transcription of PIF4 and shows that overall PIF4 transcription may be determined by the relative levels of PIF4 to BZR1 protein. We also investigated the role of MED25 in transcriptional regulation of PIF4. We showed that PIF4 expression is upregulated in med25 mutant at ambient temperature, while PIF4 expression is super induced compared to WT at warm temperature. However, this upregulation of PIF4 expression is uncoupled from induction of PIF4 target gene YUCCA8 and from the elongation response. The regulation of PIF4 gene transcription and regulation of PIF4 target genes by PIF4 has an additional layer, as histone modifications at these promoters also become part of the equation. We provide evidence that MED25 may actually recruit histone modifying activity for PIF4 target genes. In last chapter, we describe a novel strategy by which plants can be transformed with a single construct to obtain overexpression of the transgene and silencing of an endogenous target gene of interest. The concept of a transgene containing an aimiRNA could be useful for simultaneous manipulation of several gene activities, which could be an important tool for plant biotechnology.</p

RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.)

RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium ssp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialisation

HISTONE DEACETYLASE 9 stimulates auxin-dependent thermomorphogenesis in Arabidopsis thaliana by mediating H2A.Z depletion

Many plant species respond to unfavorable high ambient temperatures by adjusting their vegetative body plan to facilitate cooling. This process is known as thermomorphogenesis and is induced by the phytohormone auxin. Here, we demonstrate that the chromatin-modifying enzyme HISTONE DEACETYLASE 9 (HDA9) mediates thermomorphogenesis but does not interfere with hypocotyl elongation during shade avoidance. HDA9 is stabilized in response to high temperature and mediates histone deacetylation at the YUCCA8 locus, a rate-limiting enzyme in auxin biosynthesis, at warm temperatures. We show that HDA9 permits net eviction of the H2A.Z histone variant from nucleosomes associated with YUCCA8, allowing binding and transcriptional activation by PHYTOCHROME INTERACTING FACTOR 4, followed by auxin accumulation and thermomorphogenesis

Transcriptional Feedback in Plant Growth and Defense by PIFs, BZR1, HY5, and MYC Transcription Factors

Growth of Arabidopsis is controlled by the activity of a set of bHLH and bZIP transcription factors of which phytochrome interacting factor4 (PIF4), BRASSINAZOLE-RESISTANT 1 (BZR1), and elongated hypocotyl 5 (HY5) have been most extensively studied. Defense responses are controlled by a set of MYC transcription factors of which MYC2 is best characterized. Moreover, hundreds of additional proteins (here named co-factors) have been identified which (in)directly may affect the expression or activity of these TFs. Thus, regulation of expression of genes encoding these co-factors becomes an integral part of understanding the molecular control of growth and defense. Here, we review RNA-seq data related to PIF, BZR1, HY5, or MYC activity, which indicate that 125 co-factor genes affecting PIFs, HY5, BZR1, or MYCs are themselves under transcriptional control by these TFs, thus revealing potential feedback regulation in growth and defense. The transcriptional feedback on co-factor genes related to PIF4, BZR1, and MYC2 by PIFs, BZR1, or MYCs, mostly results in negative feedback on PIF4, BZR1, or MYC2 activity. In contrast, transcription feedback on co-factor genes for HY5 by HY5 mostly results in positive feedback on HY5 activity. PIF4 and BZR1 exert a balanced regulating of photoreceptor-gene expression, whose products directly or indirectly affect PIF4, HY5, and MYC2 protein stability as a function of light. Growth itself is balanced by both multiple positive and multiple negative feedback on PIF4 and BZR1 activity. The balance between growth and defense is mostly through direct cross-regulation between HY5 and MYC2 as previously described, but also through potential transcriptional feedback on co-factor genes for MYC2 by PIF4, BZR1, and HY5 and through transcriptional feedback of co-factors for PIF4 and BZR1 by MYC2. The interlocking feed-forward and feed-backward transcriptional regulation of PIF4, BZR1, HY5, and MYC2 co-factors is a signature of robust and temporal control of signaling related to growth and defense

Functional intron-derived miRNAs and host-gene expression in plants

Background: Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC. Results: The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure. Conclusion: The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations

Functional intron-derived miRNAs and host-gene expression in plants

Background: Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC. Results: The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure. Conclusion: The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations

The Mediator complex subunit MED25 interacts with HDA9 and PIF4 to regulate thermomorphogenesis

Thermomorphogenesis is, among other traits, characterized by enhanced hypocotyl elongation due to the induction of auxin biosynthesis genes like YUCCA8 by transcription factors, most notably PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Efficient binding of PIF4 to the YUCCA8 locus under warmth depends on HISTONE DEACETYLASE 9 (HDA9) activity, which mediates histone H2A.Z depletion at the YUCCA8 locus. However, HDA9 lacks intrinsic DNA-binding capacity, and how HDA9 is recruited to YUCCA8, and possibly other PIF4-target sites, is currently not well understood. The Mediator complex functions as a bridge between transcription factors bound to specific promoter sequences and the basal transcription machinery containing RNA polymerase II. Mutants of Mediator component Mediator25 (MED25) exhibit reduced hypocotyl elongation and reduced expression of YUCCA8 at 27°C. In line with a proposed role for MED25 in thermomorphogenesis in Arabidopsis (Arabidopsis thaliana), we demonstrated an enhanced association of MED25 to the YUCCA8 locus under warmth and interaction of MED25 with both PIF4 and HDA9. Genetic analysis confirmed that MED25 and HDA9 operate in the same pathway. Intriguingly, we also showed that MED25 destabilizes HDA9 protein. Based on our findings, we propose that MED25 recruits HDA9 to the YUCCA8 locus by binding to both PIF4 and HDA9

Histone Deacetylase 9 stimulates auxin-dependent thermomorphogenesis in Arabidopsis thaliana by mediating H2A.Z depletion

Many plant species respond to unfavorable high ambient temperatures by adjusting their vegetative body plan to facilitate cooling. This process is known as thermomorphogenesis and is induced by the phytohormone auxin. Here, we demonstrate that the chromatin-modifying enzyme HISTONE DEACETYLASE 9 (HDA9) mediates thermomorphogenesis but does not interfere with hypocotyl elongation during shade avoidance. HDA9 is stabilized in response to high temperature and mediates histone deacetylation at the YUCCA8 locus, a rate-limiting enzyme in auxin biosynthesis, at warm temperatures. We show that HDA9 permits net eviction of the H2A.Z histone variant from nucleosomes associated with YUCCA8, allowing binding and transcriptional activation by PHYTOCHROME INTERACTING FACTOR 4, followed by auxin accumulation and thermomorphogenesis

HISTONE DEACETYLASE 9 stimulates auxin-dependent thermomorphogenesis in Arabidopsis thaliana by mediating H2A.Z depletion

Many plant species respond to unfavorable high ambient temperatures by adjusting their vegetative body plan to facilitate cooling. This process is known as thermomorphogenesis and is induced by the phytohormone auxin. Here, we demonstrate that the chromatin-modifying enzyme HISTONE DEACETYLASE 9 (HDA9) mediates thermomorphogenesis but does not interfere with hypocotyl elongation during shade avoidance. HDA9 is stabilized in response to high temperature and mediates histone deacetylation at the YUCCA8 locus, a rate-limiting enzyme in auxin biosynthesis, at warm temperatures. We show that HDA9 permits net eviction of the H2A.Z histone variant from nucleosomes associated with YUCCA8, allowing binding and transcriptional activation by PHYTOCHROME INTERACTING FACTOR 4, followed by auxin accumulation and thermomorphogenesis