KNOTTED1-like Homeobox Genes Research Articles

PagKNAT2/6b regulates tension wood formation and gravitropism by targeting cytokinin metabolism.

Tension wood is a specialized xylem tissue associated with gravitropism in angiosperm trees. However, few regulators of tension wood formation have been identified. The molecular mechanisms underpinning tension wood formation remain elusive. Here, we report that a Populus KNOTTED-like homeobox gene, PagKNAT2/6b, is involved in tension wood formation and gravity response. Transgenic poplar plants overexpressing PagKNAT2/6b displayed more sensitive gravitropism than controls, as indicated by increased stem curvature. Microscopic examination revealed greater abundance of fibre cells with a gelatinous cell wall layer (G-layer) and asymmetric growth of secondary xylem in PagKNAT2/6b overexpression lines. Conversely, PagKNAT2/6b dominant repression plants exhibited decreased tension wood formation and reduced response to gravity stimulation. Moreover, sensitivity to gravity stimulation showed a negative relationship with development stage. Expression of genes related to growth and senescence was affected in PagKNAT2/6b transgenic plants. More importantly, transcription activation and electrophoretic mobility shift assays suggested that PagKNAT2/6b promotes the expression of cytokinin metabolism genes. Consistently, cytokinin content was increased in PagKNAT2/6b overexpression plants. Therefore, PagKNAT2/6b is involved in gravitropism and tension wood formation, likely via modulation of cytokinin metabolism.

Genome-wide analysis of TOPLESS/TOPLESS-RELATED co-repressors and functional characterization of BnaA9.TPL regulating the embryogenesis and leaf morphology in rapeseed

TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins belong to the Groucho (Gro)/Tup1 family co-repressors and act as broad co-repressors that modulate multiple phytohormone signalling pathways and various developmental processes in plant. However, TPL/TPR co-repressors so far are poorly understood in the rapeseed, one of the world-wide important oilseed crops. In this study, we comprehensively characterized eighteen TPL/TPR genes into five groups in the rapeseed genome. Members of TPL/TPR1/TPR4 and TPR2/TPR3 had close evolutionary relationship, respectively. All TPL/TPRs had similar expression patterns and encode conserved protein domain. In addition, we demonstrated that BnaA9.TPL interacted with all known plant repression domain (RD) sequences, which were distributed in non-redundant 24,238 (22.6 %) genes and significantly enriched in transcription factors in the rapeseed genome. These transcription factors were largely co-expressed with the TPL/TPR genes and involved in diverse pathway, including phytohormone signal transduction, protein kinases and circadian rhythm. Furthermore, BnaA9.TPL was revealed to regulate apical embryonic fate by interaction with Bna.IAA12 and suppression of PLETHORA1/2. BnaA9.TPL was also identified to regulate leaf morphology by interaction with Bna.AS1 (Asymmetric leaves 1) and suppression of KNOTTED-like homeobox genes and YABBY5. These data not only suggest the rapeseed TPL/TPRs play broad roles in different processes, but also provide useful information to uncover more TPL/TPR-mediated control of plant development in rapeseed.

Genomic analysis of a spontaneous unifoliate mutant reveals gene candidates associated with compound leaf development in Vigna unguiculata [L] Walp

Molecular mechanisms which underpin compound leaf development in some legumes have been reported, but there is no previous study on the molecular genetic control of compound leaf formation in Vigna unguiculata (cowpea), an important dryland legume of African origin. In most studied species with compound leaves, class 1 KNOTTED-LIKE HOMEOBOX genes expressed in developing leaf primordia sustain morphogenetic activity, allowing leaf dissection and the development of leaflets. Other genes, such as, SINGLE LEAFLET1 in Medicago truncatula and Trifoliate in Solanum lycopersicum, are also implicated in regulating compound leaf patterning. To set the pace for an in-depth understanding of the genetics of compound leaf development in cowpea, we applied RNA-seq and whole genome shotgun sequence datasets of a spontaneous cowpea unifoliate mutant and its trifoliate wild-type cultivar to conduct comparative reference-based gene expression, de novo genome-wide isoform switch, and genome variant analyses between the two genotypes. Our results suggest that genomic variants upstream of LATE ELONGATED HYPOCOTYL and down-stream of REVEILLE4, BRASSINOSTERIOD INSENSITIVE1 and LATERAL ORGAN BOUNDARIES result in down-regulation of key components of cowpea circadian rhythm central oscillator and brassinosteroid signaling, resulting in unifoliate leaves and brassinosteroid-deficient-like phenotypes. We have stated hypotheses that will guide follow-up studies expected to provide more insights.

Characterization of the KNOTTED1-like HOMEOBOX gene family in kiwifruit and functional analysis of AcKNOX11 related to plant growth, flowering, and melatonin-mediated germination inhibition

KNOX (KNOTTED1-like HOMEOBOX) plays essential roles in extensive plant developmental processes, although information on the KNOX gene has not been documented in kiwifruit (Actinidia chinensis). Here, we identified 19 KNOX gene (AcKNOX) members from the kiwifruit genome. The AcKNOX could be divided into three categories, and most members contain four conserved domains. AcKNOX promoters contain abundant CREs that respond to hormonal and stress cues. AcKNOX genes express in multiple tissues, and some of these genes respond to salt stress. It has been found in this study that AcKNOX11, selectively expressed in flower bud and shoot, activated during the lateral bud development and could be evoked by ABA and melatonin treatments. The AcKNOX11 protein located in the nucleus can be predicted to interact with other protein partners. Functional analysis revealed that AcKNOX11 delayed flowering, reduced plant height, altered leaf shape, and inhibited seed germination by ABA-melatonin crosstalk in Arabidopsis. This first thorough analysis of the AcKNOX genes will provide a more in-depth understanding of AcKNOX genes in kiwifruit and lay important theoretical basis for future functional dissection.

Identification and expression analysis of the KNOX genes during organogenesis and stress responseness in Camellia sinensis (L.) O. Kuntze.

Tea plant (Camellia sinensis L.), whose leaves are the major reproductive organs, has been cultivated and consumed widely for its economic and health benefits. The Knotted1-like Homeobox (KNOX) proteins play significant roles in leaf morphology formation and development. However, the functions of KNOX proteins in tea plants are still unknown. Here, 11 CsKNOX genes from the tea plants were cloned and divided into Class I, II, and KNATM clades based on their protein sequences. These 11 CsKNOX genes were mapped on 8 out of 15 tea plant chromosomes, all localized in the nucleus. Specific spatiotemporal expression patterns of CsKNOX genes were found in various tissues and different development periods of buds, flowers, and roots of tea plants. Meanwhile, transcript levels of CsKNOX in tea leaves were strongly correlated with the accumulation of flavan-3-ols and proanthocyanidins. It was found that most of the CsKNOX genes could respond to drought, salt, cold, and exogenous MeJA and GA3 by analysis of transcriptomics data and promoter elements. The protein interaction analysis showed that CsKNOX could cooperate with CsAS1 and other critical functional proteins. In conclusion, this research provided the basic information for the functions of the CsKNOX family during organogenesis and stress response in tea plants, which was necessary for further functional characterization verification.

Genome-Wide Classification and Evolutionary Analysis of the KNOX Gene Family in Plants

The Knotted1-like homeobox (KNOX) gene family plays a pivotal role in regulating meristem activity, organ differentiation, and cell meristematic identity. However, there has been a lack of large-scale, systematic, and comprehensive comparative analyses to explore their expression patterns and evolutionary mechanisms. In this study, a total of 1425 KNOX genes were identified across 118 plant species. The result showed that higher plants exhibited a significantly higher abundance of KNOX genes compared to lower plants. Phylogenetic analysis revealed that all KNOX genes can be divided into two classes (class I and II) and evolved independently after species differentiation. An analysis of gene duplication or loss showed that gene loss was more common than gene duplication in lower plants within the KNOX gene family. These findings suggest that gene loss in the KNOX gene family occurs after events such as whole-genome duplication (WGD) or whole-genome triplication (WGT). In addition, conserved motif analysis was also conducted to uncover the evolutionary trajectories of KNOX genes. We found that three motifs (M1, M2, and M4) were present in nearly all KNOX genes, while four novel motifs (M7–M10) were lost in lower plants but present in higher plants. Moreover, the loss of certain motifs in the KNOX genes was also observed in higher plants, indicating sequence divergence in KNOX genes throughout evolution. To understand the expression patterns of KNOX genes, a gene expression pattern analysis was performed in A. thaliana and O. sativa. The results showed that class I KNOX genes exhibit conserved high expression in stems, suggesting their potential similar biological roles across different plant species and the conservation of their functions during evolution. Additionally, we analyzed the KNOX genes in the Citrus genus and closely related species, and we found that the number of KNOX genes evolved at a slower rate in these species, indicating a relatively conservative nature. In conclusion, this study provides valuable resources for the investigation of KNOX gene family evolution and function in plants.

AtHSPR functions in gibberellin-mediated primary root growth by interacting with KNAT5 and OFP1 in Arabidopsis.

AtHSPR forms a complex with KNAT5 and OFP1 to regulate primary root growth through GA-mediated root meristem activity. KNAT5-OFP1 functions as a negative regulator of AtHSPR in response to GA. Plant root growth is modulated by gibberellic acid (GA) signaling and depends on root meristem maintenance. ARABIDOPSIS THALIANA HEAT SHOCK PROTEIN-RELATED (AtHSPR) is a vital regulator of flowering time and salt stress tolerance. However, little is known about the role of AtHSPR in the regulation of primary root growth. Here, we report that athspr mutant exhibits a shorter primary root compared to wild type and that AtHSPR interacts with KNOTTED1-LIKE HOMEOBOX GENE 5 (KNAT5) and OVATE FAMILY PROTEIN 1 (OFP1). Genetic analysis showed that overexpression of KNAT5 or OFP1 caused a defect in primary root growth similar to that of the athspr mutant, but knockout of KNAT5 or OFP1 rescued the short root phenotype in the athspr mutant by altering root meristem activity. Further investigation revealed that KNAT5 interacts with OFP1 and that AtHSPR weakens the inhibition of GIBBERELLIN 20-OXIDASE 1 (GA20ox1) expression by the KNAT5-OFP1 complex. Moreover, root meristem cell proliferation and root elongation in 35S::KNAT5athspr and 35S::OFP1athspr seedlings were hypersensitive to GA3 treatment compared to the athspr mutant. Together, our results demonstrate that the AtHSPR-KNAT5-OFP1 module regulates root growth and development by impacting the expression of GA biosynthetic gene GA20ox1, which could be a way for plants to achieve plasticity in response to the environment.

Genome-Wide Identification of the KNOX Gene Family in Japanese Apricot (Prunus mume Sieb. et Zucc.) and Functional Characterization of PmKNAT2 Genes.

The Knotted1-like Homeobox gene is crucial for plant morphological development and growth. Physicochemical characteristics, phylogenetic relationships, chromosomal localization, cis-acting elements, and tissue-specific expression patterns of the 11 PmKNOX genes found in the Japanese apricot genome in this study were examined. Proteins of 11 PmKNOX were soluble proteins with isoelectric points between 4.29 and 6.53, molecular masses between 15.732 and 44.011 kDa, and amino acid counts between 140 and 430. The identified PmKNOX gene family was split into three subfamilies by jointly constructing the phylogenetic tree of KNOX proteins in Japanese apricot and Arabidopsis thaliana. Combined outcomes of the analyzed conserved motifs and gene structures of the 11 PmKNOX genes from the same subfamily displayed comparable gene structure and motif patterns. The 11 PmKNOX members were distributed across six chromosomes, while two sets of PmKNOX genes were found to be collinear. Analysis of the 2000 bp promoter upstream of the coding region of the PmKNOX gene revealed that most PmKNOX genes might be involved in the physiological metabolism, growth and development processes of plants. The PmKNOX gene expression profile revealed that these genes were expressed at varying levels in different tissues, and most of them were linked to the meristems of leaf and flower buds, suggesting that PmKNOX may be involved in plants' apical meristems. In Arabidopsis thaliana, functional validation of PmKNAT2a and PmKNAT2b revealed that these two genes might be involved in regulating leaf and stem development. In addition to laying the groundwork for future research on the function of these genes, understanding the evolutionary relationships between members of the PmKNOX gene family provides opportunities for future breeding in Japanese apricots.

Prunus Knotted-like Genes: Genome-Wide Analysis, Transcriptional Response to Cytokinin in Micropropagation, and Rootstock Transformation.

Knotted1-like homeobox (KNOX) transcription factors are involved in plant development, playing complex roles in aerial organs. As Prunus species include important fruit tree crops of Italy, an exhaustive investigation of KNOX genes was performed using genomic and RNA-seq meta-analyses. Micropropagation is an essential technology for rootstock multiplication; hence, we investigated KNOX transcriptional behavior upon increasing 6-benzylaminopurine (BA) doses and the effects on GF677 propagules. Moreover, gene function in Prunus spp. was assessed by Gisela 6 rootstock transformation using fluorescence and peach KNOX transgenes. Based on ten Prunus spp., KNOX proteins fit into I-II-M classes named after Arabidopsis. Gene number, class member distribution, and chromosome positions were maintained, and exceptions supported the diversification of Prunus from Cerasus subgenera, and that of Armeniaca from the other sections within Prunus. Cytokinin (CK) cis-elements occurred in peach and almond KNOX promoters, suggesting a BA regulatory role in GF677 shoot multiplication as confirmed by KNOX expression variation dependent on dose, time, and interaction. The tripled BA concentration exacerbated stress, altered CK perception genes, and modified KNOX transcriptions, which are proposed to concur in in vitro anomalies. Finally, Gisela 6 transformation efficiency varied (2.6-0.6%) with the genetic construct, with 35S:GFP being more stable than 35S:KNOPE1 lines, which showed leaf modification typical of KNOX overexpression.

Measurements of the number of specified and unspecified cells in the shoot apical meristem during a plastochron in rice (Oryza sativa) reveal the robustness of cellular specification process in plant development.

The shoot apical meristem (SAM) is composed of a population of stem cells giving rise to the aboveground parts of plants. It maintains itself by controlling the balance of cell proliferation and specification. Although knowledge of the mechanisms maintaining the SAM has been accumulating, the processes of cellular specification to form leaves and replenishment of unspecified cells in the SAM during a plastochron (the time interval between which two successive leaf primordia are formed) is still obscure. In this study, we developed a method to quantify the number of specified and unspecified cells in the SAM and used it to elucidate the dynamics of cellular specification in the SAM during a plastochron in rice. OSH1 is a KNOX (KNOTTED1-like homeobox) gene in rice that is expressed in the unspecified cells in the SAM, but not in specified cells. Thus, we could visualize and count the nuclei of unspecified cells by fluorescent immunohistochemical staining with an anti-OSH1 antibody followed by fluorescein isothiocyanate detection. By double-staining with propidium iodide (which stains all nuclei) and then overlaying the images, we could also detect and count the specified cells. By using these measurements in combination with morphological observation, we defined four developmental stages of SAM that portray cellular specification and replenishment of unspecified cells in the SAM during a plastochron. In addition, through the analysis of mutant lines with altered size and shape of the SAM, we found that the number of specified cells destined to form a leaf primordium is not affected by mild perturbations of meristem size and shape. Our study highlights the dynamism and flexibility in stem cell maintenance in the SAM during a plastochron and the robustness of plant development.

Genome-Wide Identification and Expression Pattern Analysis of KNOX Gene Family in Orchidaceae.

The establishment of lateral organs and subsequent plant architecture involves factors intrinsic to the stem apical meristem (SAM) from which they are derived. KNOTTED1-LIKE HOMEOBOX (KNOX) genes are a family of plant-specific homeobox transcription factors that especially act in determining stem cell fate in SAM. Although KNOXs have been studied in many land plants for decades, there is a dearth of knowledge on KNOX’s role in Orchidaceae, the largest and most diverse lineage of flowering plants. In this study, a total of 32 putative KNOX genes were identified in the genomes of five orchid species and further designated into two classes (Class I and Class II) based on phylogenetic relationships. Sequence analysis showed that most orchid KNOX proteins retain four conserved domains (KNOX1, KNOX2, ELK, and Homeobox_KN). Comparative analysis of gene structure showed that the exon–intron structure is conserved in the same clade but most orchids exhibited longer intron, which may be a unique feature of Orchidaceae. Cis-elements identified in the promoter region of orchid KNOXs were found mostly enriched in a function of light responsiveness, followed by MeJA and ABA responsiveness, indicative of their roles in modulating light and phytohormones. Collinear analysis unraveled a one-to-one correspondence among KNOXs in orchids, and all KNOX genes experienced strong purifying selection, indicating the conservation of this gene family has been reinforced across the Orchidaceae lineage. Expression profiles based on transcriptomic data and real-time reverse transcription–quantitative PCR (RT-qPCR) revealed a stem-specific expression of KNOX Class I genes and a broader expression pattern of Class II genes. Taken together, our results provided a comprehensive analysis to uncover the underlying function of KNOX genes in Orchidaceae.

The PagKNAT2/6b-PagBOP1/2a Regulatory Module Controls Leaf Morphogenesis in Populus.

Leaf morphogenesis requires precise regulation of gene expression to achieve organ separation and flat-leaf form. The poplar KNOTTED-like homeobox gene PagKNAT2/6b could change plant architecture, especially leaf shape, in response to drought stress. However, its regulatory mechanism in leaf development remains unclear. In this work, gene expression analyses of PagKNAT2/6b suggested that PagKNAT2/6b was highly expressed during leaf development. Moreover, the leaf shape changes along the adaxial-abaxial, medial-lateral, and proximal-distal axes caused by the mis-expression of PagKNAT2/6b demonstrated that its overexpression (PagKNAT2/6b OE) and SRDX dominant repression (PagKNAT2/6b SRDX) poplars had an impact on the leaf axial development. The crinkle leaf of PagKNAT2/6b OE was consistent with the differential expression gene PagBOP1/2a (BLADE-ON-PETIOLE), which was the critical gene for regulating leaf development. Further study showed that PagBOP1/2a was directly activated by PagKNAT2/6b through a novel cis-acting element “CTCTT”. Together, the PagKNAT2/6b-PagBOP1/2a module regulates poplar leaf morphology by affecting axial development, which provides insights aimed at leaf shape modification for further improving the drought tolerance of woody plants.

Genome-Wide Analysis of KNOX Transcription Factors and Expression Pattern of Dwarf-Related KNOX Genes in Pear.

KNOTTED1-like homeobox (KNOX) transcription factors (TFs) belonging to the homeobox TF family play important roles in plant growth, development, and responses to abiotic and biotic stress. However, little information is available on KNOX TF in pear (Pyrus). In this study, 19 PbKNOXs TFs were re-identified in pear (Pyrus bretschneideri Rehd.). Phylogenetic analysis revealed that the TFs were clustered into three groups with 10 conserved motifs, some of which were group- or subgroup-specific, implying that they are important for the functions of the KNOX in these clades. PbKNM1 and PbKNM2 are KNM (encodes a MEINOX domain but not a homeodomain) genes identified in pear for the first time. KNOX genes in Pyrus and Malus were closely related, and a collinear relationship among PbKNOX genes in Pyrus and Malus was observed. Analysis of the expression patterns of PbKNOX genes in different tissues, at various growth stages, and in response to abiotic and biotic stress revealed that PbKNOXs are involved in plant growth and development. Our comparative transcriptional analysis of dwarf mutant varieties revealed that genes belonging to class I are highly expressed compared with genes in other classes. Analysis of the expression of PbKNOX genes in the hybrid offspring of vigorous and dwarf varieties revealed that PbKNOX genes were highly expressed in the vigorous offspring and weakly expressed in the dwarf offspring. These findings provide new insight into the function of KNOX TFs in pear and will aid future studies of dwarf fruit trees.

Evolutionary Relationships and Divergence of KNOTTED1-Like Family Genes Involved in Salt Tolerance and Development in Cotton (Gossypium hirsutum L.).

The KNOX (KNOTTED1-like homeobox) transcription factors play an important role in leaf, shoot apical meristem and seed development and respond to biotic and abiotic stresses. In this study, we analyzed the diversity and evolutionary history of the KNOX gene family in the genome of tetraploid cotton (Gossypium hirsutum). Forty-four putative KNOX genes were identified. All KNOX genes from seven higher plant species were classified into KNOXI, KNOXII, and KNATM clades based on a phylogenetic analysis. Chromosomal localization and collinearity analysis suggested that whole-genome duplication and a polyploidization event contributed to the expansion of the cotton KNOX gene family. Analyses of expression profiles revealed that the GhKNOX genes likely responded to diverse stresses and were involved in cotton growth developmental processes. Silencing of GhKNOX2 enhanced the salt tolerance of cotton seedlings, whereas silencing of GhKNOX10 and GhKNOX14 reduced seedling tolerance to salt stress. Silencing of GhSTM3 influenced the cotton flowering time and plant development. These findings clarify the evolution of the cotton KNOX gene family and provide a foundation for future functional studies of KNOX proteins in cotton growth and development and response to abiotic stresses.

Genome wide identification of StKNOX gene family and characterization of their expression in Solanum tuberosum

The KNOTTED1-LIKE HOMEOBOX (KNOX) transcription factors (TF) play an important role in growth and developmental stages in potato. However, only few reports are available regarding the KNOX gene family in potato. In this study, 17 KNOX genes were detected in the potato genome. The gene structure, protein characteristics, promoter analysis, synthetic regions, gene duplication, gene expression in developmental stages, and chromosomal location were investigated. Based on their phylogenetic relationships, the StKNOX family members were categorized into three classes. Analysis of gene expression showed that the tissue expression patterns of StKNOX genes in the same group were similar but varied among groups. Analysis of transcription factor binding site (TFBS) and microRNA in the promoter region and coding sequence of StKNOX genes revealed the presence of putative TFBS and 49 StmiRNAs. Moreover, variable frequencies of TFSB and miRNA in StKNOX genes could indicate that these genes control different developmental stages and are involved in complex regulatory mechanisms. Our results showed that some members of Class II (StKNOX14) have the highest number of TFBS and are highly expressed in 19 potato tissues. Our findings showed that these genes could be involved in the regulation of plant growth and tuber development in S.tuberosum . The results of this study will be useful in further investigations on the functional role of potato StKNOX genes. • KNOX proteins are homeodomain transcription factors that have a critical role in morphogenesis. • Some members of KNAT2/6 (KNOX6/7/8) have the highest number of TFBS and are highly expressed in plant roots. • KNOX genes could be involved in regulation of plant growth and tuber development in S.tuberosum.

Knox homologs shoot meristemless (STM) and KNAT6 are epistatic to CLAVATA3 (CLV3) during shoot meristem development in Arabidopsis thaliana.

InArabidopsis,the genes SHOOT MERISTEMLESS(STM) andCLAVATA3 (CLV3) antagonistically regulate shoot meristem development.STMis essential for both development and maintenance of the meristem, asstmmutants fail to develop a shoot meristem.CLV3, on the other hand, negatively regulates meristem proliferation, andclv3mutants possess an enlarged shoot meristem. Genetic interaction studies revealed thatstmandclv3 dominantly suppress each other's phenotypes. STM works in conjunction withits closely related homologue KNOTTED1-LIKE HOMEOBOX GENE 6 (KNAT6)to promote meristem development and organ separation, asstmknat6double mutants fail to form shoot meristem and produce a fused cotyledon. In this study, we show thatclv3fails topromoteshoot meristem formation instm-1background if we also remove KNAT6.stm-1knat6 clv3triple mutants result in shoot meristem termination and produce fused cotyledons similar tostmknat6double mutant. Notably, the stm-1 knat6 and stm-1 knat6 clv3 alleles lack tissue in the presumed region of SAM that is a novel phenotype reported in Arabidopsis mutants.stm-1knat6 clv3also showed reduced inflorescence size as compared toclv3single orstmclv3double mutants. In contrast to previously published data, these data suggest thatSTM and KNAT6 are redundantly required for the vegetative SAM, but insufficient for the inflorescence meristem.

RNA-seq between asexual archeospores and meiosis-related conchospores in Neopyropia yezoensis using Smart-seq2.

In the life cycle of Neopyropia yezoensis, a potential model system for marine macroalgae, both asexual archeospores and meiosis-related conchospores develop into thalli (gametophyte). To understand this special life phenomenon in macroalgae, we picked out the two kinds of spores (10-30 cells in each sample) and conducted RNA-seq using Smart-seq2. Comparative analysis showed that light capture and carbon fixation associated differentially expressed genes (DEGs) were upregulated in archeospores, thus indicating that archeospores are in a state of rapid vegetative growth. In conchospores, protein synthesis and degradation, especially molecular chaperone, associated DEGs were up-regulated, indicating that complex life activities might be occurring in conchospores. There were 68 genes related to DNA replication and repair expressed in conchospores, showing that active DNA replication might occur in conchospores. Moreover, we found that one conchospore specifically expressed DEG (py04595: DNA helicase) only in diploid stages (conchocelis, sporangial filament) and three archeospores specifically expressed DEGs only in haploid stages (thalli). These molecular level results indicated that conchospores were closer to diploid, and might be the meiotic mother cells of N.yezoensis. In addition, we found that the knotted-like homeobox gene (PyKNOX), which might relate to the transition of gametophyte from sporophyte, was only expressed insporophyte generation but not expressed in conchospores, archeospores and thalli, indicating the morphogenesis of gametophyte sin N.yezoensis might require the inactivation of PyKNOX.

Protocol for screening and expression studies of T-DNA and tagging-based insertional knox mutants in Arabidopsis thaliana.

The online version contains supplementary material available at 10.1007/s13205-021-02868-8.

MdKNOX19, a class II knotted-like transcription factor of apple, plays roles in ABA signalling/sensitivity by targeting ABI5 during organ development

The ABI5 transcription factor, which is a core component of the ABA signaling pathway, affects various plant processes, including seed development and germination and responses to environmental cues. The knotted1-like homeobox (KNOX) transcription factor has crucial functions related to plant development, including the regulation of various hormones. In this study, an ABA-responsive KNOX gene, MdKNOX19, was identified in apple (Malus domestica). The overexpression of MdKNOX19 increased the ABA sensitivity of apple calli, resulting in a dramatic up-regulation in the transcription of the Arabidopsis ABI5-like MdABI5 gene. Additionally, MdKNOX19 overexpression in Micro-Tom adversely affected fruit size and seed yield as well as enhanced ABA sensitivity and up-regulated SlABI5 transcription during seed germination and early seedling development. An examination of MdKNOX19-overexpressing Arabidopsis plants also revealed severe defects in seed development and up-regulated expression of ABA-responsive genes. Furthermore, we further confirmed that MdKNOX19 binds directly to the MdABI5 promoter to activate expression. Our findings suggest MdKNOX19 is a positive regulator of ABI5 expression, and the conserved module MdKNOX19-MdABI5-ABA may contribute to organ development.

Characterization of the KNOTTED1-LIKE HOMEOBOX (KNOX) gene family in Pinus pinaster Ait.

KNOTTED1-LIKE HOMEOBOX (KNOX) genes are a family of plant-specific homeobox transcription factors with important roles in plant development that have been classified into two subfamilies with differential expression domains and functions. Studies in angiosperms have shown that class I members are related to the maintenance of meristem homeostasis and leaf development, whereas class II members promote differentiation of tissues and organs. However, little is known about its diversification and function in gymnosperms. By combining PCR-based detection and transcriptome data analysis, we identified four class I and two class II KNOX genes in Pinus pinaster. Expression analyses showed that class I members were mainly expressed in meristematic regions and differentiating tissues, with practically no expression in lateral organs, whereas expression of class II members was restricted to lateral organs. Furthermore, overexpression of P. pinaster KNOX genes in Arabidopsis thaliana caused similar phenotypic effects to those described for their angiosperms counterparts. This is the first time to our knowledge that functional analyses of class II members are reported in a conifer species. These results suggest a high conservation of the KNOX gene family throughout seed plants, as the functional differentiation of both subfamilies observed in angiosperms might be partially conserved in gymnosperms.