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Cloning and Functional Analysis of ThActin Gene in Tetrastigma hemsleyanum from Huaiyu Mountain
HONGSenrong, CHENDandan, CHAShuli, WUKetong, WUJingyan
Chin Agric Sci Bull ›› 2026, Vol. 42 ›› Issue (21) : 188-197.
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Abbreviation (ISO4): Chin Agric Sci Bull
Editor in chief: Yulong YIN
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Cloning and Functional Analysis of ThActin Gene in Tetrastigma hemsleyanum from Huaiyu Mountain
Actin is a type of structural protein that is ubiquitous and highly conserved in eukaryotes. As a major component of the cytoskeleton, it participates in fundamental biological processes such as cell division, cytokinesis, and endocytosis, and plays a critical role in stress resistance. To investigate the ThActin gene of Tetrastigma hemsleyanum from Huaiyu Mountain, we screened and cloned this gene based on transcriptome data, constructed the pCambia2301 overexpression vector, and performed a transient transformation subcellular localization assay in leaf epidermal cells of tobacco (Nicotiana tabacum W38). Subsequently, the construct was transformed into tobacco to generate ThActin transgenic tobacco plants, and low-temperature stress treatment was applied to characterize the functions of the transgenic lines. The results showed that the full-length of ThActin gene was 1131 bp. Transient transformation subcellular localization analysis in tobacco leaf epidermal cells revealed that the ThActin protein localized to the cytoplasm and nucleus. Genetic transformation experiments in tobacco demonstrated that under low-temperature stress, compared with wild-type tobacco, ThActin-overexpressing tobacco exhibited significantly reduced contents of hydrogen peroxide, superoxide anion and malondialdehyde, markedly increased activities of antioxidant enzymes (SOD, POD and CAT), and significantly elevated levels of osmotic adjustment substances (proline, soluble sugar and soluble protein). These findings indicated that overexpression of ThActin enhances plant cold tolerance. This study provides a reference for elucidating the low-temperature stress tolerance mechanism of Tetrastigma hemsleyanum from Huaiyu Mountain and breeding its cold-resistant cultivars.
Tetrastigma hemsleyanum from Huaiyu Mountain / ThActin / gene cloning / subcellular localization / gene overexpression / low temperature stress / antioxidant enzymes / osmotic adjustment substances
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ACTIN DEPOLYMERIZING FACTORs (ADFs) regulate the organization and dynamics of actin microfilaments (AFs) by cleavage and depolymerization of AFs. The Arabidopsis thaliana genome encodes 11 ADF genes grouped into 4 subclasses. Subclass I ADFs, ADF1, −2, −3, and −4, are expressed in all vegetative tissues and are reportedly involved in the regulation of plant growth, and abiotic and biotic stress responses. Furthermore, the nuclear localization of ADF4 is seemingly important in disease response. Here, we present data that indicate a previously unknown regulatory role of subclass I ADFs in the regulation of leaf senescence. ADF4 knockout mutants (adf4) and transgenic lines in which the expression of all members of subclass I ADFs was downregulated (ADF1-4Ri) showed acceleration of both dark-induced and age-dependent leaf senescence. Upregulation of the expression of senescence-associated genes, such as SAG13, SGR1, PPH, and WRKY53, occurred earlier in adf4 and ADF1-4Ri lines than in wild type. Examination of the expression of subclass I ADFs genes during age-dependent leaf senescence revealed a reduced expression of ADF4 but not expressions of other subclass I members. Additionally, we showed that nuclear localization of ADF4 was important for regulating leaf senescence. Altogether, our data indicate that subclass I ADFs, particularly ADF4, play an important role in the regulation of leaf senescence.
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In Arabidopsis, the actin gene family comprises eight expressed and two non-expressed ACTIN (ACT) genes. Of the eight expressed isoforms, ACT2, ACT7, and ACT8 are differentially expressed in vegetative tissues and may perform specific roles in development. Using tobacco mesophyll protoplasts, we previously demonstrated that actin-dependent clustering of chloroplasts around the nucleus prior to cell division ensures unbiased chloroplast inheritance. Here, we report that actin-dependent chloroplast clustering in Arabidopsis mesophyll protoplasts is defective in act7 mutants, but not act2-1 or act8-2. ACT7 expression was upregulated during protoplast culture whereas ACT2 and ACT8 expression did not substantially change. In act2-1, ACT7 expression increased in response to loss of ACT2, whereas in act7-1, neither ACT2 nor ACT8 expression changed appreciably in response to the absence of ACT7. Semi-quantitative immunoblotting revealed increased actin concentrations during culture, although total actin in act7-1 was only two-thirds that of wild-type or act2-1 after 96 h culture. Over-expression of ACT2 and ACT8 under control of ACT7 regulatory sequences restored normal levels of chloroplast clustering. These results are consistent with a requirement for ACT7 in actin-dependent chloroplast clustering due to reduced levels of actin protein and gene induction in act7 mutants, rather than strong functional specialization of the ACT7 isoform.
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Root hairs develop as long extensions from root epidermal cells. After the formation of an initial bulge at the distal end of the epidermal cell, the root hair structure elongates by tip growth. Because root hairs are not surrounded by other cells, root hair formation provides an excellent system for studying the highly complex process of plant cell growth. Pharmacological experiments with actin filament-interfering drugs have provided evidence that the actin cytoskeleton is an important factor in the establishment of cell polarity and in the maintenance of the tip growth machinery at the apex of the growing root hair. However, there has been no genetic evidence to directly support this assumption. We have isolated an Arabidopsis mutant, deformed root hairs 1 (der1), that is impaired in root hair development. The DER1 locus was cloned by map-based cloning and encodes ACTIN2 (ACT2), a major actin of the vegetative tissue. The three der1 alleles develop the mutant phenotype to different degrees and are all missense mutations, thus providing the means to study the effect of partially functional ACT2. The detailed characterization of the der1 phenotypes revealed that ACT2 is not only involved in root hair tip growth, but is also required for correct selection of the bulge site on the epidermal cell. Thus, the der1 mutants are useful tools to better understand the function of the actin cytoskeleton in the process of root hair formation.
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During plant growth and development, the phytohormone auxin induces a wide array of changes that include cell division, cell expansion, cell differentiation, and organ initiation. It has been suggested that the actin cytoskeleton plays an active role in the elaboration of these responses by directing specific changes in cell morphology and cytoarchitecture. Here we demonstrate that the promoter and the protein product of one of the Arabidopsis vegetative actin genes, ACT7, are rapidly and strongly induced in response to exogenous auxin in the cultured tissues of Arabidopsis. Homozygous act7-1 mutant plants were slow to produce callus tissue in response to hormones, and the mutant callus contained at least two to three times lower levels of ACT7 protein than did the wild-type callus. On the other hand, a null mutation in ACT2, another vegetative actin gene, did not significantly affect callus formation from leaf or root tissue. Complementation of the act7-1 mutants with the ACT7 genomic sequence restored their ability to produce callus at rates similar to those of wild-type plants, confirming that the ACT7 gene is required for callus formation. Immunolabeling of callus tissue with actin subclass-specific antibodies revealed that the predominant ACT7 is coexpressed with the other actin proteins. We suggest that the coexpression, and probably the copolymerization, of the abundant ACT7 with the other actin isovariants in cultured cells may facilitate isovariant dynamics well suited for cellular responses to external stimuli such as hormones.
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We have previously shown that the wheat (Triticum aestivum) TaADF gene expression level is correlated with the plants capacity to tolerate freezing. Sequence analysis revealed that this gene encodes a protein homologous to members of the actin-depolymerizing factor (ADF)/cofilin family. We report here on the characterization of the recombinant TaADF protein. Assays for ADF activity showed that TaADF is capable of sequestering actin, preventing nucleotide exchange, and inducing actin depolymerization. In vitro phosphorylation studies showed that TaADF is a substrate for a wheat 52-kD kinase. The activity of this kinase is modulated by low temperature during the acclimation period. Western-blot analyses revealed that TaADF is expressed only in cold-acclimated Gramineae species and that the accumulation level is much higher in the freezing-tolerant wheat cultivars compared with the less tolerant ones. This accumulation was found to be regulated by a factor(s) encoded by a gene(s) located on chromosome 5A, the chromosome most often found to be associated with cold hardiness. The induction of an active ADF during cold acclimation and the correlation with an increased freezing tolerance suggest that the protein may be required for the cytoskeletal rearrangements that may occur upon low temperature exposure. These remodelings might be important for the enhancement of freezing tolerance.
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Calcium (Ca2+) plays important roles in plant growth, development, and signal transduction. It is a vital nutrient for plant physical design, such as cell wall and membrane, and also serves as a counter-cation for biochemical, inorganic, and organic anions, and more particularly, its concentration change in cytosol is a ubiquitous second messenger in plant physiological signaling in responses to developmental and environmental stimuli. Actin cytoskeleton is well known for its importance in cellular architecture maintenance and its significance in cytoplasmic streaming and cell division. In plant cell system, the actin dynamics is a process of polymerization and de-polymerization of globular actin and filamentous actin and that acts as an active regulator for calcium signaling by controlling calcium evoked physiological responses. The elucidation of the interaction between calcium and actin dynamics will be helpful for further investigation of plant cell signaling networks at molecular level. This review mainly focuses on the recent advances in understanding the interaction between the two aforementioned signaling components in two well-established model systems of plant, guard cell, and pollen.
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