Expression of a Chlorophyll <i>b</i> Reductase Gene from <i>Zoysia japonica</i> Causes Changes in Leaf Color and Chlorophyll Morphology in <i>Agrostis stolonifera</i>

The NYC-like (NOL) enzyme is considered as an essential enzyme for chlorophyll b degradation, which catalyzes the formation of 7-hydroxymethyl chlorophyll a from chlorophyll b. The ZjNOL gene was cloned from Zoysia japonica with a completed coding sequence of 981-bp in length, encoding 326 amino acids. ZjNOL was localized on the stroma side of the thylakoid membrane, and co-localized with ZjNYC in the chloroplasts. Multiple photoregulatory elements and hormone regulatory elements were identified in the promoter region of the ZjNOL gene, and the expression level of the ZjNOL gene was dramatically up-regulated in senescence leaves, which were regulated by a variety of plant hormones. ZjNOL’s ectopic expression in creeping bentgrass produced yellow leaves, thicker cortex, and smaller vascular column cells. Additionally, transgenic plants exhibited morphological alterations in their chloroplast structure, and the number of grana and thylakoids per grana stack reduced dramatically. Transgenic plants also had a lower photosynthetic rate and Fm/Fv than the control. The transgenic plants displayed a decreased chlorophyll content and a greater rate of ion leakage. The properties and activities of ZjNOL will serve as a foundation for future research into gene functions and regulatory processes

Expression of a Hydroxycinnamoyl-CoA Shikimate/Quinate Hydroxycinnamoyl Transferase 4 Gene from <i>Zoysia japonica</i> (<i>ZjHCT4</i>) Causes Excessive Elongation and Lignin Composition Changes in <i>Agrostis stolonifera</i>

Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) is considered to be an essential enzyme for regulating the biosynthesis and composition of lignin. To investigate the properties and function of ZjHCT4, the ZjHCT4 gene was cloned from Zoysia japonica with a completed coding sequence of 1284-bp in length, encoding 428 amino acids. The ZjHCT4 gene promoter has several methyl jasmonate (MeJA) response elements. According to analysis of expression patterns, it was up-regulated by MeJA, GA3 (Gibberellin), and SA (Salicylic acid), and down-regulated by ABA (Abscisic acid). Ectopic ZjHCT4 expression in creeping bentgrass causes excessive plant elongation. In addition, the content of G-lingnin and H-lingnin fell in transgenic plants, whereas the level of S-lingnin increased, resulting in a considerable rise in the S/G unit ratio. Analysis of the expression levels of lignin-related genes revealed that the ectopic expression of ZjHCT4 altered the expression levels of a number of genes involved in the lignin synthesis pathway. Simultaneously, MeJA, SA, GA3, IAA, BR (Brassinosteroid), and other hormones were dramatically enhanced in transgenic plants relative to control plants, whereas ABA concentration was significantly decreased. Expression of ZjHCT4 impacted lignin composition and plant growth via altering the phenylpropionic acid metabolic pathway and hormone response, as revealed by transcriptome analysis. HCTs may influence plant lignin composition and plant development by altering hormone content. These findings contributed to a deeper comprehension of the lignin synthesis pathway and set the stage for further investigation and application of the HCTs gene

Table_1_Overexpression of abscisic acid-insensitive gene ABI4 from Medicago truncatula, which could interact with ABA2, improved plant cold tolerance mediated by ABA signaling.XLSX

ABI4 is considered an important transcription factor with multiple regulatory functions involved in many biological events. However, its role in abiotic stresses, especially low-temperature-induced stress, is poorly understood. In this study, the MtABI4 gene was derived from M. truncatula, a widely used forage grass. Analysis of subcellular localization indicated that ABI4 was localized in the nucleus. Identification of expression characteristics showed that ABI4 was involved in the regulatory mechanisms of multiple hormones and could be induced by the low temperature. IP-MS assay revealed that MtABI4 protein could interact with xanthoxin dehydrogenase protein (ABA2). The two-hybrid yeast assay and the biomolecular fluorescence complementarity assay further supported this finding. Expression analysis demonstrated that overexpression of MtABI4 induced an increase in ABA2 gene expression both in M. truncatula and Arabidopsis, which in turn increased the ABA level in transgenic plants. In addition, the transgenic lines with the overexpression of MtABI4 exhibited enhanced tolerance to low temperature, including lower malondialdehyde content, electrical conductivity, and cell membrane permeability, compared with the wide-type lines after being cultivated for 5 days in 4°C. Gene expression and enzyme activities of the antioxidant system assay revealed the increased activities of SOD, CAT, MDHAR, and GR, and higher ASA/DHA ratio and GSH/GSSG ratio in transgenic lines. Additionally, overexpression of ABI4 also induced the expression of members of the Inducer of CBF expression genes (ICEs)-C-repeat binding transcription factor genes(CBFs)-Cold regulated genes (CORs) low-temperature response module. In summary, under low-temperature conditions, overexpression of ABI4 could enhance the content of endogenous ABA in plants through interactions with ABA2, which in turn reduced low-temperature damage in plants. This provides a new perspective for further understanding the molecular regulatory mechanism of plant response to low temperature and the improvement of plant cold tolerance.</p

Image_1_Overexpression of abscisic acid-insensitive gene ABI4 from Medicago truncatula, which could interact with ABA2, improved plant cold tolerance mediated by ABA signaling.TIF

ABI4 is considered an important transcription factor with multiple regulatory functions involved in many biological events. However, its role in abiotic stresses, especially low-temperature-induced stress, is poorly understood. In this study, the MtABI4 gene was derived from M. truncatula, a widely used forage grass. Analysis of subcellular localization indicated that ABI4 was localized in the nucleus. Identification of expression characteristics showed that ABI4 was involved in the regulatory mechanisms of multiple hormones and could be induced by the low temperature. IP-MS assay revealed that MtABI4 protein could interact with xanthoxin dehydrogenase protein (ABA2). The two-hybrid yeast assay and the biomolecular fluorescence complementarity assay further supported this finding. Expression analysis demonstrated that overexpression of MtABI4 induced an increase in ABA2 gene expression both in M. truncatula and Arabidopsis, which in turn increased the ABA level in transgenic plants. In addition, the transgenic lines with the overexpression of MtABI4 exhibited enhanced tolerance to low temperature, including lower malondialdehyde content, electrical conductivity, and cell membrane permeability, compared with the wide-type lines after being cultivated for 5 days in 4°C. Gene expression and enzyme activities of the antioxidant system assay revealed the increased activities of SOD, CAT, MDHAR, and GR, and higher ASA/DHA ratio and GSH/GSSG ratio in transgenic lines. Additionally, overexpression of ABI4 also induced the expression of members of the Inducer of CBF expression genes (ICEs)-C-repeat binding transcription factor genes(CBFs)-Cold regulated genes (CORs) low-temperature response module. In summary, under low-temperature conditions, overexpression of ABI4 could enhance the content of endogenous ABA in plants through interactions with ABA2, which in turn reduced low-temperature damage in plants. This provides a new perspective for further understanding the molecular regulatory mechanism of plant response to low temperature and the improvement of plant cold tolerance.</p