The Maize PI/GLO Ortholog Zmm16/sterile tassel silky ear1 Interacts with the Zygomorphy and Sex Determination Pathways in Flower Development

In monocots and eudicots, B class function specifies second and third whorl floral organ identity as described in the classic ABCE model. Grass B class APETALA3/DEFICIENS orthologs have been functionally characterized; here, we describe the positional cloning and characterization of a maize (Zea mays) PISTILLATA/GLOBOSA ortholog Zea mays mads16 (Zmm16)/sterile tassel silky ear1 (sts1). We show that, similar to many eudicots, all the maize B class proteins bind DNA as obligate heterodimers and positively regulate their own expression. However, sts1 mutants have novel phenotypes that provide insight into two derived aspects of maize flower development: carpel abortion and floral asymmetry. Specifically, we show that carpel abortion acts downstream of organ identity and requires the growth-promoting factor grassy tillers1 and that the maize B class genes are expressed asymmetrically, likely in response to zygomorphy of grass floral primordia. Further investigation reveals that floral phyllotactic patterning is also zygomorphic, suggesting significant mechanistic differences with the well-characterized models of floral polarity. These unexpected results show that despite extensive study of B class gene functions in diverse flowering plants, novel insights can be gained from careful investigation of homeotic mutants outside the core eudicot model species.     

Functional analysis of NLP genes from Botrytis elliptica

We functionally analysed two Nep1-like protein (NLP) genes from Botrytis elliptica (a specialized pathogen of lily), encoding proteins homologous to the necrosis and ethylene-inducing protein (NEP1) from Fusarium oxysporum. Single gene replacement mutants were made for BeNEP1 and BeNEP2 , providing the first example of transformation and successful targeted mutagenesis in this fungus. The virulence of both mutants on lily leaves was not affected. BeNEP1 and BeNEP2 were individually expressed in the yeast Pichia pastoris , and the necrosis-inducing activity was tested by infiltration of both proteins into leaves of several monocots and eudicots. Necrotic symptoms developed on the eudicots tobacco, Nicotiana benthamiana and Arabidopsis thaliana , and cell death was induced in tomato cell suspensions. No necrotic symptoms developed on leaves of the monocots rice, maize and lily. These results support the hypothesis that the necrosis-inducing activity of NLPs is limited to eudicots. We conclude that NLPs are not essential virulence factors and they do not function as host-selective toxins for B. elliptica .     

Analysis of the Petunia TM6 MADS box gene reveals functional divergence within the DEF/AP3 lineage

Antirrhinum majus DEFICIENS (DEF) and Arabidopsis thaliana APETALA3 (AP3) MADS box proteins are required to specify petal and stamen identity. Sampling of DEF/AP3 homologs revealed two types of DEF/AP3 proteins, euAP3 and TOMATO MADS BOX GENE6 (TM6), within core eudicots, and we show functional divergence in Petunia hybrida euAP3 and TM6 proteins. Petunia DEF (also known as GREEN PETALS [GP]) is expressed mainly in whorls 2 and 3, and its expression pattern remains unchanged in a blind (bl) mutant background, in which the cadastral C-repression function in the perianth is impaired. Petunia TM6 functions as a B-class organ identity protein only in the determination of stamen identity. Atypically, Petunia TM6 is regulated like a C-class rather than a B-class gene, is expressed mainly in whorls 3 and 4, and is repressed by BL in the perianth, thereby preventing involvement in petal development. A promoter comparison between DEF and TM6 indicates an important change in regulatory elements during or after the duplication that resulted in euAP3- and TM6-type genes. Surprisingly, although TM6 normally is not involved in petal development, 35S-driven TM6 expression can restore petal development in a def (gp) mutant background. Finally, we isolated both euAP3 and TM6 genes from seven solanaceous species, suggesting that a dual euAP3/TM6 B-function system might be the rule in the Solanaceae.     

Use of genomic history to improve phylogeny and understanding of births and deaths in a gene family

Polyploidy events have played an important role in the evolution of angiosperm genomes. Here, we demonstrate how genomic histories can increase phylogenetic resolution in a gene family, specifically the expansin superfamily of cell wall proteins. There are 36 expansins in Arabidopsis and 58 in rice. Traditional sequence-based phylogenetic trees yield poor resolution below the family level. To improve upon these analyses, we searched for gene colinearity (microsynteny) between Arabidopsis and rice genomic segments containing expansin genes. Multiple rounds of genome duplication and extensive gene loss have obscured synteny. However, by simultaneously aligning groups of up to 10 potentially orthologous segments from the two species, we traced the history of 49 out of 63 expansin-containing segments back to the ancestor of monocots and eudicots. Our results indicate that this ancestor had 15-17 expansin genes, each ancestral to an extant clade. Some clades have strikingly different growth patterns in the rice and Arabidopsis lineages, with more than half of all rice expansins arising from two ancestral genes. Segmental duplications, most of them part of polyploidy events, account for 12 out of 21 new expansin genes in Arabidopsis and 16 out of 44 in rice. Tandem duplications explain most of the rest. We were also able to estimate a minimum of 28 gene deaths in the Arabidopsis lineage and nine in rice. This analysis greatly clarifies expansin evolution since the last common ancestor of monocots and eudicots and the method should be broadly applicable to many other gene families.     

Genome-wide analysis of WOX gene family in rice, sorghum, maize, Arabidopsis and poplar

WUSCHEL-related homeobox(WOX)genes form a large gene family specifically expressed in plants.They are known to play important roles in regulating the development of plant tissues and organs by determining cell fate.Recent available whole genome sequences allow us to do more comprehensive phylogenetic analysis of the WOX genes in plants.In the present study,we identified 11 and 21 WOXs from sorghum(Sorghum bicolor)and maize(Zea mays),respectively.The 72 WOX genes from rice(Oryza sativa),sorghum,maize,Arabidopsis(Arabidopsis thaliana)and poplar(Populus trichocarpa)were grouped into three well supported clades with nine subgroups according to the amino acid sequences of their homodomains.Their phylogenetic relationship was also supported by the observation of the motifs outside the homodomain.We observed the variation of duplication events among the nine sub-groups between monocots and eudicots,for instance,more gene duplication events of WOXs within subgroup A for monocots,while,less for dicots in this subgroup.Furthermore,we observed the conserved intron/exon structural patterns of WOX genes in rice,sorghum and Arabidopsis.In addition,WUS(Wuschel)-box and EAR(the ERF-associated amphiphilic repression)-like motif were observed to be conserved among several WOX subgroups in these five plants.Comparative analysis of expression patterns of WOX genes in rice and Arabidopsis suggest that the WOX genes play conserved and various roles in plants.This work provides insights into the evolution of the WOX gene family and is useful for future research.     

Functional diversification of B MADS-box homeotic regulators of flower development: Adaptive evolution in protein-protein interaction domains after major gene duplication events

B-class MADS-box genes have been shown to be the key regulators of petal and stamen specification in several eudicot model species such as Arabidopsis thaliana, Antirrhinum majus, and Petunia hybrida. Orthologs of these genes have been found across angiosperms and gymnosperms, and it is thought that the basic regulatory function of B proteins is conserved in seed plant lineages. The evolution of B genes is characterized by numerous duplications that might represent key elements fostering the functional diversification of duplicates with a deep impact on their role in the evolution of the floral developmental program. To evaluate this, we performed a rigorous statistical analysis with B gene sequences. Using maximum likelihood and Bayesian methods, we estimated molecular substitution rates and determined the selective regimes operating at each residue of B proteins. We implemented tests that rely on phylogenetic hypotheses and codon substitution models to detect significant differences in substitution rates (DSRs) and sites under positive adaptive selection (PS) in specific lineages before and after duplication events. With these methods, we identified several protein residues fixed by PS shortly after the origin of PISTILLATA-like and APETALA3-like lineages in angiosperms and shortly after the origin of the euAP3-like lineage in core eudicots, the 2 main B gene duplications. The residues inferred to have been fixed by positive selection lie mostly within the K domain of the protein, which is key to promote heterodimerization. Additionally, we used a likelihood method that accommodates DSRs among lineages to estimate duplication dates for AP3-PI and euAP3-TM6, calibrating with data from the fossil record. The dates obtained are consistent with angiosperm origins and diversification of core eudicots. Our results strongly suggest that novel multimer formation with other MADS proteins could have been crucial for the functional divergence of B MADS-box genes. We thus propose a mechanism of functional diversification and persistence of gene duplicates by the appearance of novel multimerization capabilities after duplications. Multimer formation in different combinations of regulatory proteins can be a mechanistic basis for the origin of novel regulatory functions and a gene regulatory mechanism for the appearance of morphological innovations.     

Conserved C-terminal motifs of the Arabidopsis proteins APETALA3 and PISTILLATA are dispensable for floral organ identity function

The B-class genes APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis (Arabidopsis thaliana) and their orthologs in other species have been the focus of studies to elucidate the development of petals and stamens in angiosperm flowers. Evolutionary analysis indicates that B-class genes have undergone multiple gene duplication events in angiosperms. The resultant B-class lineages are characterized by short, conserved amino acid sequences at the extreme C-terminal end of the B-class proteins. AP3 is a member of the euAP3 lineage that contains both the euAP3 and PI-derived motifs at the C terminus. PI is a member of the PI lineage that contains the C-terminal PI motif at the C terminus. Despite conservation over a wide evolutionary distance, the function of C-terminal motifs is not well understood. In this study, we demonstrate that truncated forms of AP3 and PI, which lack the conserved C-terminal motifs, function to direct floral organ identity specification in Arabidopsis plants. By contrast, larger truncations, which remove the third putative amphipathic alpha-helix in the K domain of AP3 or PI, are nonfunctional. We conclude that the euAP3 and PI-derived motifs of AP3 and the PI motif of PI are not essential for floral organ identity function of AP3 and PI in Arabidopsis.     

Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS.

Recent genetic and molecular studies in Arabidopsis and Antirrhinum suggest that mechanisms controlling floral development are well conserved among dicotyledonous species. To assess whether similar mechanisms also operate in more distantly related monocotyledonous species, we have begun to clone homologs of Arabidopsis floral genes from maize. Here we report the characterization of two genes, designated ZAG1 and ZAG2 (for Zea AG), that were cloned from a maize inflorescence cDNA library by low stringency hybridization with the AGAMOUS (AG) cDNA from Arabidopsis. ZAG1 encodes a putative polypeptide of 286 amino acids having 61% identity with the AGAMOUS (AG) protein. Through a stretch of 56 amino acids, constituting the MADS domain, the two proteins are identical except for two conservative amino acid substitutions. The ZAG2 protein is less similar to AG, with 49% identity overall and substantially less similarity than ZAG1 outside the well-conserved MADS domain. Like AG, ZAG1 RNA accumulates early in stamen and carpel primordia. In contrast, ZAG2 expression begins later and is restricted to developing carpels. Hybridization to genomic DNA with the full-length ZAG1 cDNA under moderately stringent conditions indicated the presence of a large family of related genes. Mapping data using maize recombinant inbreds placed ZAG1 and ZAG2 near two loci that are known to affect maize flower development, Polytypic ear (Pt) and Tassel seed4 (Ts4), respectively. The ZAG1 protein from in vitro translations binds to a consensus target site that is recognized by the AG protein. These data suggest that maize contains a homolog of the Arabidopsis floral identity gene AG and that this gene is conserved in sequence and function.     

Phylogenomic analyses of the BARREN STALK1/LAX PANICLE1 (BA1/LAX1) genes and evidence for their roles during axillary meristem development

The diversity of plant architectural form is largely determined by the extent and duration of axillary meristem (AM) derived lateral growth. The orthologous basic helix-loop-helix (bHLH) proteins maize BARREN STALK1 (BA1) and rice LAX PANICLE1 (LAX1) are essential for the formation of AMs during vegetative development and all lateral structures during inflorescence development, but whether BA1/LAX1 co-orthologs exist outside of the grass family is unclear. Here, we present Bayesian phylogenetic evidence of a well-supported BA1/LAX1 clade comprised monocots and eudicots, estimating an origin for the lineage at least near the base of flowering plants. Genomic analyses in Arabidopsis, papaya, medicago, rice, sorghum, and maize indicate that BA1/LAX1 genes reside in syntenic regions, although there has also been a complex pattern of gene duplication and loss during the diversification of the angiosperm clade. BA1/LAX1 mRNA expression coincided with the initiation of leaves and associated AMs in the vegetative meristems of broccoli, medicago, and papaya implicating a role for the lineage in the formation of AMs in eudicots as well as monocots. Expression on the adaxial surface of lateral inflorescence structures was conserved in all sampled flowering plants, whereas mRNA expression in leaves of Arabidopsis, broccoli, and papaya also links BA1/LAX1 co-orthologs with roles in regulating leaf development, possibly as a downstream target of auxin regulating genes. Together these data point to roles for BA1/LAX1 genes during AM formation, leaf, and inflorescence development in diverse flowering plants and lend support to the hypothesis that the same genetic mechanisms regulate the development of different AM types.     

A novel calcium/calmodulin-regulated kinesin-like protein is highly conserved between monocots and dicots

Recently, a novel kinesin-like protein (KCBP) that is regulated by Ca2+/calmodulin was isolated from dicot plants. A homolog of KCBP has not been reported in monocots. To determine if this motor protein is present in phylogenetically divergent flowering plants, Arabidopsis KCBP cDNA was used as a probe to screen a genomic library of maize, an evolutionarily divergent species. This screening resulted in isolation of a KCBP homolog. Comparison of the predicted amino acid sequence of the KCBP from maize (ZmKCBP), a monocot, with the previously reported KCBP sequences from dicot species showed that the amino acid sequence, domain organization, and gene structure are highly conserved between monocots and dicots. The C-terminal region of ZmKCBP, containing the motor domain and the calmodulin-binding domain, and the N-terminal tail, with a myosin tail homology region (MyTH4) and talin-like region, showed strong sequence similarity to the KCBP homolog from dicots. However, the coiled-coil region is less conserved between monocots and dicots. The ZmKCBP gene contained 22 exons and 21 introns. The location of 19 of the 21 introns of ZmKCBP is also conserved. The ZmKCBP protein is encoded by a single gene and expressed in all tissues. Affinity-purified antibody to the calmodulin-binding domain of Arabidopsis KCBP detected a protein in both the soluble and the microsomal fractions. The C-terminal region of ZmKCBP, containing the motor and calmodulin-binding domains, bound calmodulin in the presence of calcium and failed to bind in the presence of EGTA. The ZmKCBP, along with other KCBPs from dicots, was grouped into a distinct group in the C-terminal subfamily of kinesin-like proteins. These data suggest that the KCBP is ubiquitous and highly conserved in all flowering plants and the origin of KCBP predated the divergence of monocots and dicots.