ZmICE2 Regulates Stomatal Development in Maize

doi:10.11983/CBB22261

Abstract

Abstract: Plant epidermis is crucial in regulating photosynthesis, respiration, heat dissipation, and water utilization. Significant progress has been made in the study of stomatal development in dicotyledonous plants, such as Arabidopsis thaliana. Three important bHLH transcription factors (SPCH, MUTE, and FAMA) have been reported to be specifically expressed at different stages of cell division and differentiation in the stomatal lineage. They form heterodimers with another transcription factors SCRM/ICE1 and SCRM2/ICE2 to regulate the morphological transformation and changes of stomatal lineage cells across three stages of division, finally forming the stomatal complex. However, in monocots, especially in Poaceae plants such as maize (Zea mays), studies on genes regulating epidermal morphogenesis are less reported. In this study, two single-gene recessive mutants, Zmice1-1 (inducer of cbf expression1-1) and Zmice2-1, were isolated using reverse genetics approaches. Compared to the control B73, Zmice2-1 exhibited dwarfism, leaf chlorosis, reduced fertility, significantly lower stomatal density and index, disrupted arrangement of epidermal long cells, and absence of spacing between stomata. Zmice1-1 leaves gradually turned yellow from the five-leaf stage and displayed complete chlorosis at later stages. The homozygous Zmice1-1 plants are growth-arrested and sterile, but the stomatal density showed no significant difference compared to the control. Different allels of Zmice2 were obtained using CRISPR-Cas9 genome editing technology. Phenotypic identification showed that Zmice2-2 had an abnormal stomatal phenotype similar to Zmice2-1, indicating that ZmICE2 is involved in the regulation of stomatal development. Transcriptome analysis of B73 and Zmice2-1 revealed that ZmICE2 primarily regulated stomatal development by affecting cell division and differentiation, participating in the formation of maize epidermal morphology. These results contribute to a better understanding of the mechanisms of epidermal morphogenesis in maize and provide valuable genetic resources for improving crop resilience and yield traits.

Key words: maize, stomatal development, stomatal density, epidermal morphogenesis, CRISPR-Cas9 gene editing

Wenqi Zhou, Yuqian Zhou, Yongsheng Li, Haijun He, Yanzhong Yang, Xiaojuan Wang, Xiaorong Lian, Zhongxiang Liu, Zhubing Hu. ZmICE2 Regulates Stomatal Development in Maize[J]. Chinese Bulletin of Botany, 2023, 58(6): 866-881.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22261

https://www.chinbullbotany.com/EN/Y2023/V58/I6/866

Figures/Tables 9

Table 1 Primer sequences used in this study

Primer name	Forward primer (5′-3′)	Reverse primer (5′-3′)
ZmICE2	ACCCCAACAAAACCAGGAC	GTCTCTTTTTCAGCGGTCTTG
T-ice2-1	GAGGAGGACGACGACAAGAAG	GCTAATTGCTACCGAAAACGC
T-ice1-1	GCGACCCATCAACCCATAGC	CGGTGTTGATGGGTTGAAGC
T-ice2-c9	ACGAGAATGGGTGAGTGTGG	AAGAGCGAGAACATCTGCGA
GAPDH	CCATCACTGCCACACAGAAAAC	AGGAACACGGAAGGACATACCAG
RT-ZmICE2	CTTCCTCGGGCGGCGGCGGTG	ACCGTGCTGTTGGCGTTGGAG

Figure 1 Field phenotypes of Zmice1-1 and Zmice2-1 mutants at seedling stage (A)-(C) Zmice2-1 mutant showed yellow leaf phenotype and short plant type at seedling stage; (D), (E) From the five-leaf and one-heart stage, the Zmice1-1 leaves gradually began to turn yellow, and the phenotype became more obvious at the later stage; (F), (G) The plant height and ear height of Zmice2-1 and Zmice1-1 were significantly decreased compared with the control (** indicate extremely significant differences (P<0.01)). In the figure (G), since the homozygote of Zmice1-1 had not developed a female spike, the spike height was calculated as the height from the ground of the fallen 6 leaves at maturity stage. (B), (C), (D) Bars=10 cm; (A), (E) Bars=20 cm

Figure 2 Morphology and stomatal distribution of Zmice1-1 and Zmice2-1 mutants (A) Schematic diagram of stomata and epidermal cells in leaves of B73 seedlings (with one stomata spaced by one epidermal long cell in a monolinear regular arrangement); (B) Stomata and epidermal cell schema of young leaves of Zmice2-1 (there were only a few stomata in the same size field of vision, and stomatal density decreased significantly); (C) Stomata and epidermal cell pattern of Zmice1-1 young leaves (showed no significant difference from the control); (D) Epidermal cell morphology of B73; (E) The epidermal cell morphology of Zmice2-1 (in the setting stage number of stomata reduced); (F) Stomatal morphology and epidermal flat cell morphology of leaves at Zmice1-1 setting stage (with increased stomatal opening). Arrows indicate immature stomatas. Bars=20 μm

Figure 3 Stomatal density and stomatal index of B73 and Zmice mutants (A), (B) The stomatal density of the 3rd, 4th, 12th and 13th leaves was calculated, the stomatal density of Zmice2-1 was significantly lower than that of the control, while the stomatal density of Zmice1-1 showed no significant change (n=200); (C), (D) The stomatal indexes of the 3rd, 4th, 12th and 13th leaves were counted, Zmice2-1 stomatal indexes were significantly lower than that of the control, while Zmice1-1 stomatal indexes had no significant change (n=200). n=200 represented the statistical number of stomata (≥15 microscopic fields) and 3 biological replicates. ** P<0.01

Table 2 Gene code and function annotation of ZmICE2 in different species

Species	Homologous gene	The function of prediction
Arabidopsis thaliana	AT3G26744	Basic helix-loop-helix (bHLH) DNA-binding superfamily protein
Oryza sativa	LOC_Os11g32100	Inducer of CBF expression 1, putative, expressed
Brachypodium distachyum	Bradi4g17460	ICE87
Zea mays	GRMZM2G033356	Helix-loop-helix DNA-binding domain containing protein
Sorghum bicolor	Sb05g019530	Helix-loop-helix DNA-binding domain containing protein
Vitis vinifera	GSVIVG00008637001	Helix-loop-helix DNA-binding domain containing protein
V. vinifera	GSVIVG00032998001	Inducer of CBF expression 1
Populus trichocarpa	POPTR_0012s10780	ICE1; DNA binding/transcription activator/transcription factor
P. trichocarpa	POPTR_0015s11650	ICE1; DNA binding/transcription activator/transcription factor

Figure 4 Sequence alignment of amino acid sequence of ZmICE2 with its homologues and phylogenetic analysis (A) Conservative analysis of ZmICE2 protein sequences (with black indicating 100% identical amino acid sequences; dark gray means 75% the same amino acid sequences; light gray means 50% the same amino acid sequences); (B) Homologous tree analysis of ZmICE2 protein; (C) Phylogenetic tree analysis of ZmICE2 protein

Figure 5 Cluster analysis of differentially expressed genes in B73 and Zmice2-1 (A) Sample correlation analysis based on differential expression gene; (B) MA map of differentially expressed genes (each dot represents a gene); (C) Cluster analysis diagram of differentially expressed genes (each row represents a gene and each column represents a sample); (D) Boxplot of transcript expression (ZmICE2 expression decreased in Zmice2-1).

Figure 6 GO cluster of differentially expressed genes in B73 and Zmice2-1

Figure 7 Phenotype of ZmICE2 gene editing mutant (A) ZmICE2 transgenic test-tube seedlings; (B), (C) Phenotype of ZmICE2 gene editing T0 generation transgenic plant in field; (D) Phenotype of T2 generation transgenic plant in field (leaf yellowing); (E), (F) The epidermal stomatal schema of control (CK) and Zmice2-2 (Zmice2-2 lacked a large number of stomata and could not form normal stomata). Arrows indicate immature stomata. (A)-(D) Bars=10 cm; (E), (F) Bars=20 μm

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