Identification and Expression Analysis of CCCH Zinc Finger Family Genes in Oryza sativa

Abstract

Background: CCCH zinc finger proteins (OsC3Hs) are a class of transcriptional regulators that play important roles in plant development and stress responses. Although their functional significance has been widely studied in model species, comprehensive genome-wide characterization of CCCH proteins in rice (Oryza sativa) remains limited.

Methods: Using Arabidopsis CCCH proteins as references, we identified the CCCH gene family in rice and analyzed the physicochemical properties, subcellular localization, conserved structures, phylogeny, cis-regulatory elements, synteny analysis, spatiotemporal expression patterns, and expression patterns under drought, ABA, and MeJA treatments for the identified CCCH family members.

Results: The results showed that the rice CCCH family comprises 73 members, which are unevenly distributed across the 12 chromosomes. Phylogenetic analysis classified them into 11 subfamilies. Subcellular localization indicated that most members are localized in the nucleus. The upstream regions of CCCH promoters contain a large number of cis-regulatory elements related to plant hormones and biotic stress responses. Most genes respond to drought, abscisic acid (ABA), and methyl jasmonate (MeJA) treatments. OsC3H36 was highly expressed under drought, ABA, and MeJA treatments. Haplotype analysis of this gene revealed two major allelic variants (H1 and H2), with H1 predominantly found in japonica rice and associated with increased grain width and 1000-grain weight. Functional validation using a chromosome segment substitution line (CSSL1) confirmed these findings.

Conclusions: CCCH genes play important roles in rice growth, development, and stress responses. Additionally, we validated that OsC3H36 is associated with rice grain width and 1000-grain weight.

Keywords: CCCH gene family; gene expression; haplotype analysis; phenotypic analysis; rice.

Figure 1

Characteristics and comparison of CCCH-type zinc finger proteins. (A) The number of CCCH proteins and CCCH motifs in O. sativa, A. thaliana, Z. may, H. vulgare, G. max, P. virgatum, B. napus, B. rape, S. lycopersicum, M. truncatula, T. aestivum, V. vinifers, C. clementine, and P. trichocarpe. (B) The proportion of CCCH proteins containing 1, 2, 3, 4, 5, 6, or 7 CCCH motifs in O. sativa, A. thaliana, Z. may, H. vulgare, G. max, P. virgatum, B. napus, B. rape, S. lycopersicum, M. truncatula, T. aestivum, V. vinifers, C. clementine, and P. trichocarpe. (C) The number of each type of CCCH motif in O. sativa, A. thaliana, Z. may, H. vulgare, G. max, P. virgatum, B. napus, B. rape, S. lycopersicum, M. truncatula, T. aestivum, V. vinifers, C. clementine, and P. trichocarpe.

Figure 2

Phylogenetic relationships, motif composition, and domain analysis of rice CCCH genes. (A) Motifs of OsC3H proteins predicted by MEME, with different colors representing different motifs. (B) Domains of OsC3H proteins, with different colors indicating different domains.

Figure 3

Distribution of CCCH genes on chromosomes and synteny relationships in rice. Red genes represent segmental duplicated genes, while blue boxes indicate tandem duplicated genes.

Figure 4

Phylogenetic relationships and synteny analysis of 14 Species. (A) Phylogenetic relationships among O. sativa, A. thaliana, Zea may, H. vulgare, G. max, S. lycopersicum, P. virgatum, T. aestivum, B. napus, B. rape, M. truncatula, C. clementine, V. vinifera, and P. trichocarpe, along with the number of homologous genes shared with rice from the other 13 species. (B) Synteny analysis between O. sativa and A. thaliana. The gray lines indicate collinear blocks between O. sativa and A. thaliana, and the red lines colinear gene pairs between O. sativa and A. thaliana. (C) Synteny analysis between O. sativa and Z. may. The gray lines indicate collinear blocks between O. sativa and Z. may, and the red lines colinear gene pairs between O. sativa and Z. may.

Figure 5

Number of cis-regulatory elements in rice OsC3Hs. Different font colors represent various types of elements. Blue, green, and red indicate stress response, tissue-specific expression, and plant growth regulators, respectively.

Figure 6

Expression profiles of OsC3Hs in different tissues.

Figure 7

Expression levels in shoot and root of rice after drought, MeJA, and ABA treatments. (A) Expression levels in shoot. (B) Expression levels in root.

Figure 8

OsC3H36 haplotype analysis. (A) The gene structure of OsC3H36. The coding region is indicated by a black box; 3′ UTR and 5′ UTR are indicated by white boxes. (B) Haplotype analysis of expression level and three phenotypes (grain length, grain width, grain weight). Different low-case letters above columns indicate statistical differences at p < 0.05. Red represents haplotype H1, and blue represents haplotype H2.

Figure 9

Phenotypes of 9311 and substitution lines. (A) Grain length (left) and grain width (right) of 9311 and substitution lines. (B) Grain length, grain width, and grain weight of 9311 and substitution lines. Different low-case letters above columns indicate statistical differences at p < 0.05. Red represents haplotype H1, and blue represents haplotype H2.