Lijun An

Zinc Finger Protein 6 (ZFP6) regulates trichome initiation by integrating gibberellin and cytokinin signaling in Arabidopsis thaliana

The Arabidopsis trichome is a model system for studying cell development, cell differentiation and the cell cycle in plants. Our previous studies have shown that the ZINC FINGER PROTEIN5 (ZFP5) controls shoot maturation and epidermal cell fate through GA signaling in Arabidopsis. We have identified a novel C2H2 zinc finger protein ZINC FINGER PROTEIN 6 (ZFP6) which plays a key role in regulating trichome development in Arabidopsis. Overexpression of ZFP6 results in ectopic trichomes on carpels and other inflorescence organs. Gain- and loss-of-function analyses have shown that the zfp6 mutant exhibits a reduced number of trichomes in sepals of flowers, cauline leaves, lateral branch and main inflorescence stems in comparison to wild-type plants. Molecular and genetic analyses suggest that ZFP6 functions upstream of GIS, GIS2, ZFP8, ZFP5 and key trichome initiation regulators GL1 and GL3.We reveal that ZFP6 and ZFP5 mediate the regulation of trichome initiation by integrating GA and cytokinin signaling in Arabidopsis. These findings provide new insights into the molecular mechanism of plant hormone control of epidermal trichome patterning through C2H2 transcriptional factors.

Zinc Finger Protein5 Is Required for the Control of Trichome Initiation by Acting Upstream of Zinc Finger Protein8 in Arabidopsis

Arabidopsis (Arabidopsis thaliana) trichome development is a model system for studying cell development, cell differentiation, and the cell cycle. Our previous studies have shown that the GLABROUS INFLORESCENCE STEMS (GIS) family genes, GIS, GIS2, and ZINC FINGER PROTEIN8 (ZFP8), control shoot maturation and epidermal cell fate by integrating gibberellins (GAs) and cytokinin signaling in Arabidopsis. Here, we show that a new C2H2 zinc finger protein, ZFP5, plays an important role in controlling trichome cell development through GA signaling. Overexpression of ZFP5 results in the formation of ectopic trichomes on carpels and other inflorescence organs. zfp5 loss-of-function mutants exhibit a reduced number of trichomes on sepals, cauline leaves, paraclades, and main inflorescence stems in comparison with wild-type plants. More importantly, it is found that ZFP5 mediates the regulation of trichome initiation by GAs. These results are consistent with ZFP5 expression patterns and the regional influence of GA on trichome initiation. The molecular analyses suggest that ZFP5 functions upstream of GIS, GIS2, ZFP8, and the key trichome initiation regulators GLABROUS1 (GL1) and GL3. Using a steroid-inducible activation of ZFP5 and chromatin immunoprecipitation experiments, we further demonstrate that ZFP8 is the direct target of ZFP5 in controlling epidermal cell differentiation.

A zinc finger protein gene ZFP5 integrates phytohormone signaling to control root hair development in Arabidopsis

Although root hair development in Arabidopsis thaliana has been extensively studied, it remains unknown whether the zinc finger proteins, the largest family of transcription factors in plants, are involved in this process. Here we report that the C2H2 zinc finger protein ZINC FINGER PROTEIN 5 (ZFP5) is a key regulator of root hair initiation and morphogenesis in Arabidopsis. ZFP5 is mainly expressed in root and preferentially in root hair cells. Using both zfp5 mutants and ZFP5 RNAi lines, we show that reduction in ZFP5 function leads to fewer and much shorter root hairs compared to wild-type. Genetic and molecular experiments demonstrate that ZFP5 exerts its effect on root hair development by directly promoting expression of the CAPRICE (CPC) gene. Furthermore, we show that ZFP5 expression is induced by cytokinin, and that ZFP5 mediates cytokinin and ethylene effects on the formation and growth of root hairs. These results suggest that ZFP5 integrates various plant hormone cues to control root epidermal cell development in Arabidopsis.

Progress on trichome development regulated by phytohormone signaling

Trichomes are specialized structures that develop from epidermal cells in the aerial parts of plants, and are an excellent model system to study all aspects of cell differentiation including cell fate determination, cell cycle regulation, cell polarity and cell expansion. The development of the trichome is a process of integration of both external signals and endogenous developmental programs. During recent years, molecular analysis of trichome development at different stages has been well studied, and through the mutant phenotypes and the function of corresponding genes, the underlying mechanism has been revealed in a first glimpse. This paper offers a mini-view on this integration process with emphasis on the effects of plant hormone signaling on trichome development in plants through GLABROUS INFLORESCENCE STEMS (GIS) family and subfamily genes.

GLABROUS INFLORESCENCE STEMS (GIS) is Required for Trichome Branching Through Gibberellic Acid Signaling in Arabidopsis

Cell differentiation generally corresponds to the cell cycle, typically forming a non-dividing cell with a unique differentiated morphology, and Arabidopsis trichome is an excellent model system to study all aspects of cell differentiation. Although gibberellic acid is reported to be involved in trichome branching in Arabidopsis, the mechanism for such signaling is unclear. Here, we demonstrated that GLABROUS INFLORESCENCE STEMS (GIS) is required for the control of trichome branching through gibberellic acid signaling. The phenotypes of a loss-of-function gis mutant and an overexpressor showed that GIS acted as a repressor to control trichome branching. Our results also show that GIS is not required for cell endoreduplication, and our molecular and genetic study results have shown that GIS functions downstream of the key regulator of trichome branching, STICHEL (STI), to control trichome branching through the endoreduplication-independent pathway. Furthermore, our results also suggest that GIS controls trichome branching in Arabidopsis through two different pathways and acts either upstream or downstream of the negative regulator of gibbellic acid signaling SPINDLY (SPY).

ZFP5 encodes a functionally equivalent GIS protein to control trichome initiation

The Arabidopsis thaliana trichome development is a model system for understanding various aspects of plant cell development and differentiation. The C2H2 zinc finger proteins GIS, GIS2, and ZFP8 play important roles in controlling trichome initiation. In our recent study, we reported that a new C2H2 zinc finger protein, ZINC FINGER PROTEIN 5 (ZFP5), controls trichome cell development through GA signaling. ZFP5 acts upstream of GIS gene family and key trichome initiation regulators, and ZFP8 is the direct target gene of ZFP5. Here we show that ZFP5 encodes a protein functionally equivalent to GIS and GIS2 in controlling trichome initiation. Furthermore, similar to GIS2, ZFP5 is not involved in trichome branching.

Genetic Interactions Reveal that Specific Defects of Chloroplast Translation are Associated with the Suppression of var2‐Mediated Leaf Variegation

Arabidopsis thaliana L. yellow variegated (var2) mutant is defective in a chloroplast FtsH family metalloprotease, AtFtsH2/VAR2, and displays an intriguing green and white leaf variegation. This unique var2-mediated leaf variegation offers a simple yet powerful tool for dissecting the genetic regulation of chloroplast development. Here, we report the isolation and characterization of a new var2 suppressor gene, SUPPRESSOR OF VARIEGATION8 (SVR8), which encodes a putative chloroplast ribosomal large subunit protein, L24. Mutations in SVR8 suppress var2 leaf variegation at ambient temperature and partially suppress the cold-induced chlorosis phenotype of var2. Loss of SVR8 causes unique chloroplast rRNA processing defects, particularly the 23S-4.5S dicistronic precursor. The recovery of the major abnormal processing site in svr8 23S-4.5S precursor indicate that it does not lie in the same position where SVR8/L24 binds on the ribosome. Surprisingly, we found that the loss of a chloroplast ribosomal small subunit protein, S21, results in aberrant chloroplast rRNA processing but not suppression of var2 variegation. These findings suggest that the disruption of specific aspects of chloroplast translation, rather than a general impairment in chloroplast translation, suppress var2 variegation and the existence of complex genetic interactions in chloroplast development.

Mutations in circularly permuted GTPase family genes AtNOA1/RIF1/SVR10 and BPG2 suppress var2-mediated leaf variegation in Arabidopsis thaliana

Leaf variegation mutants constitute a unique group of chloroplast development mutants and are ideal genetic materials to dissect the regulation of chloroplast development. We have utilized the Arabidopsis yellow variegated (var2) mutant and genetic suppressor analysis to probe the mechanisms of chloroplast development. Here we report the isolation of a new var2 suppressor locus SUPPRESSOR OF VARIEGATION (SVR10). Genetic mapping and molecular complementation indicated that SVR10 encodes a circularly permuted GTPase that has been reported as Arabidopsis thaliana NITRIC OXIDE ASSOCIATED 1 (AtNOA1) and RESISTANT TO INHIBITION BY FOSMIDOMYCIN 1 (RIF1). Biochemical evidence showed that SVR10/AtNOA1/RIF1 likely localizes to the chloroplast stroma. We further demonstrate that the mutant of a close homologue of SVR10/AtNOA1/RIF1, BRASSINAZOLE INSENSITIVE PALE GREEN 2 (BPG2), can also suppress var2 leaf variegation. Mutants of SVR10 and BPG2 are impaired in photosynthesis and the accumulation of chloroplast proteins. Interestingly, two-dimensional blue native gel analysis showed that mutants of SVR10 and BPG2 display defects in the assembly of thylakoid membrane complexes including reduced levels of major photosynthetic complexes and the abnormal accumulation of a chlorophyll-protein supercomplex containing photosystem I. Taken together, our findings suggest that SVR10 and BPG2 are functionally related with VAR2, likely through their potential roles in regulating chloroplast protein homeostasis, and both SVR10 and BPG2 are required for efficient thylakoid protein complex assembly and photosynthesis.

Chloroplast translation initiation factors regulate leaf variegation and development

SVR9 and its homolog SVR9L1 encode functionally redundant chloroplast translation initiation factors essential for chloroplast and leaf development in Arabidopsis. Chloroplast development requires the coordinated expressions of nuclear and chloroplast genomes, and both anterograde and retrograde signals exist and work together to facilitate this coordination. We have utilized the Arabidopsis yellow variegated (var2) mutant as a tool to dissect the genetic regulatory network of chloroplast development. Here, we report the isolation of a new (to our knowledge) var2 genetic suppressor locus, SUPPRESSOR OF VARIEGATION9 (SVR9). SVR9 encodes a chloroplast-localized prokaryotic type translation initiation factor 3 (IF3). svr9-1 mutant can be fully rescued by the Escherichia coli IF3 infC, suggesting that SVR9 functions as a bona fide IF3 in the chloroplast. Genetic and molecular evidence indicate that SVR9 and its close homolog SVR9-LIKE1 (SVR9L1) are functionally interchangeable and their combined activities are essential for chloroplast development and plant survival. Interestingly, we found that SVR9 and SVR9L1 are also involved in normal leaf development. Abnormalities in leaf anatomy, cotyledon venation patterns, and leaf margin development were identified in svr9-1 and mutants that are homozygous for svr9-1 and heterozygous for svr9l1-1 (svr9-1 svr9l1-1/+). Meanwhile, as indicated by the auxin response reporter DR5:GUS, auxin homeostasis was disturbed in svr9-1, svr9-1 svr9l1-1/+, and plants treated with inhibitors of chloroplast translation. Genetic analysis established that SVR9/SVR9L1-mediated leaf margin development is dependent on CUP-SHAPED COTYLEDON2 activities and is independent of their roles in chloroplast development. Together, our findings provide direct evidence that chloroplast IF3s are essential for chloroplast development and can also regulate leaf development.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.