Non-cell-autonomous signaling associated with barley ALOG1 specifies spikelet meristem determinacy.

Inflorescence architecture and crop productivity are often tightly coupled in our major cereal crops. However, the underlying genetic mechanisms controlling cereal inflorescence development remain poorly understood. Here, we identified recessive alleles of barley (Hordeum vulgare L.) HvALOG1 (Arabidopsis thaliana LSH1 and Oryza G1) that produce non-canonical extra spikelets and fused glumes abaxially to the central spikelet from the upper-mid portion until the tip of the inflorescence. Notably, we found that HvALOG1 exhibits a boundary-specific expression pattern that specifically excludes reproductive meristems, implying the involvement of previously proposed localized signaling centers for branch regulation. Importantly, during early spikelet formation, non-cell-autonomous signals associated with HvALOG1 expression may specify spikelet meristem determinacy, while boundary formation of floret organs appears to be coordinated in a cell-autonomous manner. Moreover, barley ALOG family members synergistically modulate inflorescence morphology, with HvALOG1 predominantly governing meristem maintenance and floral organ development. We further propose that spatiotemporal redundancies of expressed HvALOG members specifically in the basal inflorescence may be accountable for proper patterning of spikelet formation in mutant plants. Our research offers new perspectives on regulatory signaling roles of ALOG transcription factors during the development of reproductive meristems in cereal inflorescences.

The Birth and Death of Floral Organs in Cereal Crops.

Florets of cereal crops are the basic reproductive organs that produce grains for food or feed. The birth of a floret progresses through meristem initiation and floral organ identity specification and maintenance. During these processes, both endogenous and external cues can trigger a premature floral organ death, leading to reproductive failure. Recent advances in different cereal crops have identified both conserved and distinct regulators governing the birth of a floret. However, the molecular underpinnings of floral death are just beginning to be understood. In this review, we first provide a general overview of the current findings in the field of floral development in major cereals and outline different forms of floral deaths, particularly in the Triticeae crops. We then highlight the importance of vascular patterning and photosynthesis in floral development and reproductive success and argue for an expanded knowledge of floral birth-death balance in the context of agroecology. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Anatomical insights into the vascular layout of the barley rachis: implications for transport and spikelet connection.

BACKGROUND AND AIMS: Vascular patterning is intimately related to plant form and function. Here, using barley (Hordeum vulgare) as a model, we studied the vascular anatomy of the spike-type inflorescence. The main aim of the present work was to clarify the relationship between rachis (spike axis) vasculature and spike size, to define vascular dynamics and to discuss the implications for transport capacity and its interaction with the spikelets. METHODS: We used serial transverse internode sections to determine the internode area, vascular area and number of veins along the rachis of several barley lines. KEY RESULTS: Internode area and total vascular area show a clear positive correlation with spike size, whereas the number of veins is only weakly correlated. The lateral periphery of the rachis contains large mature veins of constant size, whereas the central part is occupied by small immature veins. Spikelet-derived veins entering the rachis often merge with the immature rachis veins but never merge with the mature veins. An increase in floret fertility through the conversion of a two-rowed barley into an isogenic six-rowed line, in addition to a decrease in floret fertility owing to enhanced pre-anthesis tip degeneration caused by the mutation tip sterile 2.b (tst2.b), significantly affected vein size but had limited to no effects on the number of veins or internode area. CONCLUSIONS: The rachis vasculature is the result of a two-step process involving an initial layout followed by size adjustment according to floret fertility/spike size. The restriction of large mature vessels to the periphery and that of small immature vessels to the centre of the rachis suggests that long-distance transport and local supply to spikelets are spatially separated processes. The identification of spikelet-derived veins entering the rachis without fusing with its vasculature indicates that a vascular continuity between rachis and spikelets might be non-essential.

HOMEOBOX2, the paralog of SIX-ROWED SPIKE1/HOMEOBOX1, is dispensable for barley spikelet development.

The HD-ZIP class I transcription factor Homeobox 1 (HvHOX1), also known as Vulgare Row-type Spike 1 (VRS1) or Six-rowed Spike 1, regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic functions of HvHOX1 and HvHOX2 during spikelet development are still fragmentary. Here, we show that compared with HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of the two genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

Agroecological genetics of biomass allocation in wheat uncovers genotype interactions with canopy shade and plant size.

How plants distribute biomass among organs influences resource acquisition, reproduction and plant-plant interactions, and is essential in understanding plant ecology, evolution, and yield production in agriculture. However, the genetic mechanisms regulating allocation responses to the environment are largely unknown. We studied recombinant lines of wheat (Triticum spp.) grown as single plants under sunlight and simulated canopy shade to investigate genotype-by-environment interactions in biomass allocation to the leaves, stems, spikes, and grains. Size-corrected mass fractions and allometric slopes were employed to dissect allocation responses to light limitation and plant size. Size adjustments revealed light-responsive alleles associated with adaptation to the crop environment. Combined with an allometric approach, we demonstrated that polymorphism in the DELLA protein is associated with the response to shade and size. While a gibberellin-sensitive allelic effect on stem allocation was amplified when plants were shaded, size-dependent effects of this allele drive allocation to reproduction, suggesting that the ontogenetic trajectory of the plant affects the consequences of shade responses for allocation. Our approach provides a basis for exploring the genetic determinants underlying investment strategies in the face of different resource constraints and will be useful in predicting social behaviours of individuals in a crop community.

Dynamic Phytomeric Growth Contributes to Local Adaptation in Barley.

Vascular plants have segmented body axes with iterative nodes and internodes. Appropriate node initiation and internode elongation are fundamental to plant fitness and crop yield; however, how these events are spatiotemporally coordinated remains elusive. We show that in barley (Hordeum vulgare L.), selections during domestication have extended the apical meristematic phase to promote node initiation, but constrained subsequent internode elongation. In both vegetative and reproductive phases, internode elongation displays a dynamic proximal-distal gradient, and among subpopulations of domesticated barleys worldwide, node initiation and proximal internode elongation are associated with latitudinal and longitudinal gradients, respectively. Genetic and functional analyses suggest that, in addition to their converging roles in node initiation, flowering-time genes have been repurposed to specify the timing and duration of internode elongation. Our study provides an integrated view of barley node initiation and internode elongation and suggests that plant architecture should be recognized as a collection of dynamic phytomeric units in the context of crop adaptive evolution.

Grain yield trade-offs in spike-branching wheat can be mitigated by elite alleles affecting sink capacity and post-anthesis source activity.

Introducing variations in inflorescence architecture, such as the 'Miracle-Wheat' (Triticum turgidum convar. compositum (L.f.) Filat.) with a branching spike, has relevance for enhancing wheat grain yield. However, in the spike-branching genotypes, the increase in spikelet number is generally not translated into grain yield advantage because of reduced grains per spikelet and grain weight. Here, we investigated if such trade-offs might be a function of source-sink strength by using 385 recombinant inbred lines developed by intercrossing the spike-branching landrace TRI 984 and CIRNO C2008, an elite durum (T. durum L.) cultivar; they were genotyped using the 25K array. Various plant and spike architectural traits, including flag leaf, peduncle, and spike senescence rate, were phenotyped under field conditions for 2 consecutive years. On chromosome 5AL, we found a new modifier QTL for spike branching, branched headt3 (bht-A3), which was epistatic to the previously known bht-A1 locus. Besides, bht-A3 was associated with more grains per spikelet and a delay in flag leaf senescence rate. Importantly, favourable alleles, viz. bht-A3 and grain protein content (gpc-B1) that delayed senescence, are required to improve grain number and grain weight in the spike-branching genotypes. In summary, achieving a balanced source-sink relationship might minimize grain yield trade-offs in Miracle-Wheat.

Multilayered regulation of developmentally programmed pre-anthesis tip degeneration of the barley inflorescence.

Leaf and floral tissue degeneration is a common feature in plants. In cereal crops such as barley (Hordeum vulgare L.), pre-anthesis tip degeneration (PTD) starts with growth arrest of the inflorescence meristem dome, which is followed basipetally by the degeneration of floral primordia and the central axis. Due to its quantitative nature and environmental sensitivity, inflorescence PTD constitutes a complex, multilayered trait affecting final grain number. This trait appears to be highly predictable and heritable under standardized growth conditions, consistent with a developmentally programmed mechanism. To elucidate the molecular underpinnings of inflorescence PTD, we combined metabolomic, transcriptomic, and genetic approaches to show that barley inflorescence PTD is accompanied by sugar depletion, amino acid degradation, and abscisic acid responses involving transcriptional regulators of senescence, defense, and light signaling. Based on transcriptome analyses, we identified GRASSY TILLERS1 (HvGT1), encoding an HD-ZIP transcription factor, as an important modulator of inflorescence PTD. A gene-edited knockout mutant of HvGT1 delayed PTD and increased differentiated apical spikelets and final spikelet number, suggesting a possible strategy to increase grain number in cereals. We propose a molecular framework that leads to barley PTD, the manipulation of which may increase yield potential in barley and other related cereals.

A molecular framework for grain number determination in barley.

Flowering plants with indeterminate inflorescences often produce more floral structures than they require. We found that floral primordia initiations in barley (Hordeum vulgare L.) are molecularly decoupled from their maturation into grains. While initiation is dominated by flowering-time genes, floral growth is specified by light signaling, chloroplast, and vascular developmental programs orchestrated by barley CCT MOTIF FAMILY 4 (HvCMF4), which is expressed in the inflorescence vasculature. Consequently, mutations in HvCMF4 increase primordia death and pollination failure, mainly through reducing rachis greening and limiting plastidial energy supply to developing heterotrophic floral tissues. We propose that HvCMF4 is a sensory factor for light that acts in connection with the vascular-localized circadian clock to coordinate floral initiation and survival. Notably, stacking beneficial alleles for both primordia number and survival provides positive implications on grain production. Our findings provide insights into the molecular underpinnings of grain number determination in cereal crops.

Fast and Reproducible Matrix Deposition for MALDI Mass Spectrometry Imaging with Improved Glass Sublimation Setup.

Sublimation is one of the preferred methods of choice for matrix deposition in high spatial resolution MALDI mass spectrometry imaging (MALDI-MSI) experiments. However, reproducibility and time are the major concerns for this setup. Here we present a lab-made glass sublimator with significant improvements in fine control of the vacuum with real-time monitoring and a rapid sublimation process of only 22 min. This method yielded reproducible homogeneous matrix crystals of <1 µm on the sample surface. MALDI-MSI was performed in tissue sections of barley inflorescence meristems at 15 µm spatial resolution, thus demonstrating its efficiency. Overall, we believe these simple yet effective new modifications can be easily adapted to the standard glass sublimation devices to achieve highly reproducible matrix deposition for high spatial resolution MALDI-MSI.

Exploring the trade-off between individual fitness and community performance of wheat crops using simulated canopy shade.

The genetic heritage of wheat (Triticum spp.) crops has been shaped by millions of years of predomestication natural selection, often driven by competition among individuals. However, genetic improvements in yield potential are thought to involve selection towards reduced competitiveness, thus enhancing adaptation to the crop environment. We investigated potential trade-offs between individual plant fitness and community performance using a population of introgression lines carrying chromosome segments of wild emmer (nondomesticated) in the background of an elite durum cultivar. We focused on light as a primary factor affecting plant-plant interactions and assessed morphological and biomass phenotypes of single plants grown in mixtures under sunlight and a simulated canopy shade, and the relevance of these phenotypes for the monoculture community in the field. We found that responses to canopy shade resemble responses to high density and contribute to both the individual and the community. Stepwise regressions suggested that grain number per spike and its persistence under shade are essential attributes of productive communities, advocating their use as a breeding target during early-generation selection. Overall, multiple phenotypes attained under shade could better explain community performance. Our novel, applicable, high-throughput set-up provides new prospects for studying and selecting single-plant phenotypes in a canopy-like environment.

Genetic association of spikelet abortion with spike, grain, and shoot traits in highly-diverse six-rowed barley.

Spikelet abortion is a phenomenon where apical spikelet primordia on an immature spike abort. Regardless of the row-type, both apical and basal spikelet abortion occurs, and their extent decides the number of grain-bearing spikelets retained on the spike-thus, affecting the yield potential of barley. Reducing spikelet abortion, therefore, represents an opportunity to increase barley yields. Here, we investigated the variation for apical spikelet abortion along with 16 major spike, shoot, and grain traits in a panel of 417 six-rowed spring barleys. Our analyses showed a significantly large genotypic variation resulting in high heritability estimates for all the traits. Spikelet abortion (SA) varies from 13 to 51% depending on the genotype and its geographical origin. Among the seven spike traits, SA was negatively correlated with final spikelet number, spike length and density, while positively with awn length. This positive correlation suggests a plausible role of the rapidly growing awns during the spikelet abortion process, especially after Waddington stage 5. In addition, SA also showed a moderate positive correlation with grain length, grain area and thousand-grain weight. Our hierarchical clustering revealed distinct genetic underpinning of grain traits from the spike and shoot traits. Trait associations showed a geographical bias whereby European accessions displayed higher SA and grain and shoot trait values, whereas the trend was opposite for the Asian accessions. To study the observed phenotypic variation of SA explained by 16 other individual traits, we applied linear, quadratic, and generalized additive regression models (GAM). Our analyses of SA revealed that the GAM generally performed superior in comparison to the other models. The genetic interactions among traits suggest novel breeding targets and easy-to-phenotype "proxy-traits" for high throughput on-field selection for grain yield, especially in early generations of barley breeding programs.

Genome-Wide Association Study of Salt Tolerance-Related Traits during Germination and Seedling Development in an Intermedium-Spike Barley Collection.

Increased salinity is one of the major consequences of climatic change affecting global crop production. The early stages in the barley (Hordeum vulgare L.) life cycle are considered the most critical phases due to their contributions to final crop yield. Particularly, the germination and seedling development are sensitive to numerous environmental stresses, especially soil salinity. In this study, we aimed to identify SNP markers linked with germination and seedling development at 150 mM NaCl as a salinity treatment. We performed a genome-wide association study (GWAS) using a panel of 208 intermedium-spike barley (H. vulgare convar. intermedium (Körn.) Mansf.) accessions and their genotype data (i.e., 10,323 SNPs) using the genome reference sequence of "Morex". The phenotypic results showed that the 150 mM NaCl salinity treatment significantly reduced all recorded germination and seedling-related traits compared to the control treatment. Furthermore, six accessions (HOR 11747, HOR 11718, HOR 11640, HOR 11256, HOR 11275 and HOR 11291) were identified as the most salinity tolerant from the intermedium-spike barley collection. GWAS analysis indicated that a total of 38 highly significantly associated SNP markers under control and/or salinity traits were identified. Of these, two SNP markers on chromosome (chr) 1H, two on chr 3H, and one on chr 4H were significantly linked to seedling fresh and dry weight under salinity stress treatment. In addition, two SNP markers on chr 7H were also significantly associated with seedling fresh and dry weight but under control condition. Under salinity stress, one SNP marker on chr 1H, 5H and 7H were detected for more than one phenotypic trait. We found that in most of the accessions exhibiting the highest salinity tolerance, most of the salinity-related QTLs were presented. These results form the basis for detailed studies, leading to improved salt tolerance breeding programs in barley.

Evolution of inflorescence branch modifications in cereal crops.

Grasses are ubiquitous in our daily lives, with gramineous cereal crops such as maize, rice, and wheat constituting a large proportion of our daily staple food intake. Evolutionary forces, especially over the past ∼20 million years, have shaped grass adaptability, inflorescence architecture, and reproductive success. Here, we provide basic information on grass evolution and inflorescence structures mainly related to two inflorescence types: branched panicle- and spike-type inflorescences, the latter of which has highly modified branching. We summarize and compare known genetic pathways underlying each infloresecence type and discuss how the maize RAMOSA, rice ABERRANT PANICLE ORGANIZATION, and Triticeae COMPOSITUM pathways are regulated. Our analyses might lay the foundation for understanding species-specific gene regulatory networks that could result in improved sink capacities.

The barley mutant multiflorus2.b reveals quantitative genetic variation for new spikelet architecture.

KEY MESSAGE: Spikelet indeterminacy and supernumerary spikelet phenotypes in barley multiflorus2.b mutant show polygenic inheritance. Genetic analysis of multiflorus2.b revealed major QTLs for spikelet determinacy and supernumerary spikelet phenotypes on 2H and 6H chromosomes. Understanding the genetic basis of yield forming factors in small grain cereals is of extreme importance, especially in the wake of stagnation of further yield gains in these crops. One such yield forming factor in these cereals is the number of grain-bearing florets produced per spikelet. Wild-type barley (Hordeum vulgare L.) spikelets are determinate structures, and the spikelet axis (rachilla) degenerates after producing single floret. In contrast, the rachilla of wheat (Triticum ssp.) spikelets, which are indeterminate, elongates to produce up to 12 florets. In our study, we characterized the barley spikelet determinacy mutant multiflorus2.b (mul2.b) that produced up to three fertile florets on elongated rachillae of lateral spikelets. Apart from the lateral spikelet indeterminacy (LS-IN), we also characterized the supernumerary spikelet phenotype in the central spikelets (CS-SS) of mul2.b. Through our phenotypic and genetic analyses, we identified two major QTLs on chromosomes 2H and 6H, and two minor QTLs on 3H for the LS-IN phenotype. For, the CS-SS phenotype, we identified one major QTL on 6H, and a minor QTL on 5H chromosomes. Notably, the 6H QTLs for CS-SS and LS-IN phenotypes co-located with each other, potentially indicating that a single genetic factor might regulate both phenotypes. Thus, our in-depth phenotyping combined with genetic analyses revealed the quantitative nature of the LS-IN and CS-SS phenotypes in mul2.b, paving the way for cloning the genes underlying these QTLs in the future.

Spikelet abortion in six-rowed barley is mainly influenced by final spikelet number, with potential spikelet number acting as a suppressor trait.

The potential to increase barley grain yield lies in the indeterminate nature of its inflorescence meristem, which produces spikelets, the basic reproductive unit in grasses that are linked to reproductive success. During early reproductive growth, barley spikes pass through the maximum yield potential-a stage after which no new spikelet ridges are produced. Subsequently, spikelet abortion (SA), a phenomenon in which spikelets abort during spike growth, imposes a bottleneck for increasing the grain yield potential. Here, we studied the potential of main culm spikes by counting potential spikelet number (PSN) and final spikelet number (FSN), and computed the corresponding SA (%) in a panel of 417 six-rowed spring barleys. Our phenotypic data analyses showed a significantly large within- and across-years genotypic variation with high broad-sense heritability estimates for all the investigated traits, including SA. Asian accessions displayed the lowest SA, indicating the presence of favourable alleles that may be exploited in breeding programs. A significantly negative Pearson's product-moment correlation was observed between FSN and SA. Our path analysis revealed that PSN and FSN explain 93% of the observed phenotypic variability for SA, with PSN behaving as a suppressor trait that magnifies the effect of FSN. Based on a large set of diverse barley accessions, our results provide a deeper phenotypic understanding of the quantitative genetic nature of SA, its association with traits of high agronomic importance, and a resource for further genetic analyses.

Strategies of grain number determination differentiate barley row types.

Gaining knowledge on fundamental interactions of various yield components is crucial to improve yield potential in small grain cereals. It is well known in barley that increasing grain number greatly improves yield potential; however, the yield components determining grain number and their association in barley row types are less explored. In this study, we assessed different yield components such as potential spikelet number (PSN), spikelet survival (SSL), spikelet number (SN), grain set (GS), and grain survival (GSL), as well as their interactions with grain number by using a selected panel of two- and six-rowed barley types. Also, to analyze the stability of these interactions, we performed the study in the greenhouse and the field. From this study, we found that in two-rowed barley, grain number determination is strongly influenced by PSN rather than SSL and/or GS in both growth conditions. Conversely, in six-rowed barley, grain number is associated with SSL instead of PSN and/or GS. Thus, our study showed that increasing grain number might be possible by augmenting PSN in two-rowed genotypes, while for six-rowed genotypes SSL needs to be improved. We speculate that this disparity of grain number determination in barley row types might be due to the fertility of lateral spikelets. Collectively, this study revealed that grain number in two-rowed barley largely depends on the developmental trait, PSN, while in six-rowed barley, it mainly follows the ability for SSL.

'Spikelet stop' determines the maximum yield potential stage in barley.

Determining the grain yield potential contributed by grain number is a step towards advancing the yield of cereal crops. To achieve this aim, it is pivotal to recognize the maximum yield potential (MYP) of the crop. In barley (Hordeum vulgare L.), the MYP is defined as the maximum spikelet primordia number of a spike. Many barley studies assumed the awn primordium (AP) stage to be the MYP stage regardless of genotypes and growth conditions. From our spikelet-tracking experiments using the two-rowed cultivar Bowman, we found that the MYP stage can be different from the AP stage. Importantly, we find that the occurrence of inflorescence meristem deformation and its loss of activity coincided with the MYP stage, indicating the end of further spikelet initiation. Thus, we recommend validating the barley MYP stage with the shape of the inflorescence meristem and propose this approach (named 'spikelet stop') for MYP staging. To clarify the relevance of AP and MYP stages, we compared the MYP stage and the MYP in 27 barley accessions (two- and six-rowed accessions) grown in the greenhouse and in the field. Our results reveal that the MYP stage can be reached at various developmental stages, which greatly depend on the genotype and growth conditions. Furthermore, we propose that the MYP stage and the time to reach the MYP stage can be used to determine yield potential in barley. Based on our findings, we suggest key steps for the identification of the MYP stage in barley that may also be applied in a related crop such as wheat.

Genome-wide identification of loci modifying spike-branching in tetraploid wheat.

KEY MESSAGE: Genetic modification of spike architecture is essential for improving wheat yield. Newly identified loci for the 'Miracle wheat' phenotype on chromosomes 1AS and 2BS have significant effects on spike traits. The wheat (Triticum ssp.) inflorescence, also known as a spike, forms an unbranched inflorescence in which the inflorescence meristem generates axillary spikelet meristems (SMs) destined to become sessile spikelets. Previously, we identified the putatively causative mutation in the branched headt (bht) gene (TtBH-A1) of tetraploid wheat (T. turgidum convar. compositum (L.f.) Filat.) responsible for the loss of SM identity, converting the non-branching spike to a branched wheat spike. In the current study, we performed whole-genome quantitative trait loci (QTL) analysis using 146 recombinant inbred lines (RILs) derived from a cross between spike-branching wheat ('Miracle wheat') and an elite durum wheat cultivar showing broad phenotypic variation for spike architecture. Besides the previously found gene at the bht-A1 locus on the short arm of chromosome 2A, we also mapped two new modifier QTL for spike-branching on the short arm of chromosome 1A, termed bht-A2, and 2BS. Using biparental mapping population and GWAS in 302 diverse accessions, the 2BS locus was highly associated with coding sequence variation found at the homoeo-allele of TtBH-B1 (bht-B1). Thus, RILs that combined both bht-A1 and bht-B1 alleles showed additive genetic effects leading to increased penetrance and expressivity of the supernumerary spikelet and/or mini-spike formation.