Maize serves as a crucial cereal crop globally, yet the escalating frequency of drought stress during the reproductive phase poses a significant threat to grain yield by causing an irreversible loss in kernel number. Enhancing reproductive drought tolerance in maize requires elucidating the physiological mechanisms underlying its response to drought stress, which can then be incorporated into the development of new maize varieties through breeding programs. Additionally, innovative cultivation practices must be devised to complement these genetic improvements. In this review, the timing, duration, and severity of drought stress during the reproductive stage and their effects on maize kernel set are assessed, providing a basis for constructing a framework that links kernel setting to drought stress. Based on this framework, reproductive drought tolerance from tasseling through post-fertilization kernel establishment is subsequently examined. Evidence indicates that drought-induced fertilization failure is primarily due to delayed pollination resulting from slower silk elongation, which is caused by the loss of cell turgor and reduced carbon supply. Meanwhile, kernel abortion after fertilization is mainly triggered by carbohydrate starvation, increased ethylene emission, and the accumulation of abscisic acid (ABA). Therefore, sugar metabolism, hydraulic status, and hormone signaling collectively regulate maize’s kernel setting tolerance to drought stress in a synergistic manner. Several novel gene candidates with potential for conferring drought tolerance in maize have been identified, offering promising targets for genetic improvement through genome editing combined with targeted cultivation practices to enhance maize drought tolerance and ensure stable grain yield in future crops.

Securing maize grain yield is crucial to meet food and energy needs for the future growing population, especially under frequent drought events and elevated CO2 (eCO2) due to climate change. To maximize the kernel setting rate under drought stress is a key strategy in battling against the negative impacts. Firstly, we summarize the major limitations to leaf source and kernel sink in maize under drought stress, and identified that loss in grain yield is mainly attributed to reduced kernel set. Reproductive drought tolerance can be realized by collective contribution with a greater assimilate import into ear, more available sugars for ovary and silk use, and higher capacity to remobilize assimilate reserve. As such, utilization of CO2 fertilization by improved photosynthesis and greater reserve remobilization is a key strategy for coping with drought stress under climate change condition. We propose that optimizing planting methods and mining natural genetic variation still need to be done continuously, meanwhile, by virtue of advanced genetic engineering and plant phenomics tools, the breeding program of higher photosynthetic efficiency maize varieties adapted to eCO2 can be accelerated. Consequently, stabilizing maize production under drought stress can be achieved by securing reproductive success by harnessing CO2 fertilization.

Drought stress is detrimental to male reproduction in maize (Zea mays L.), largely through reducing the quantity and quality of pollen grains. However, transcriptional response of maize pollen grains to drought stress has not been well documented. We compared pollen gene expression for a maize hybrid (ZhongDan909) under well-watered and drought-stress conditions, based on RNA-Seq validated by quantitative real-time PCR analysis. Expression of 6424 genes and 1302 transcripts was altered in pollen grains of maize subjected to 7 days of drought during flowering. Gene Ontology annotations showed 308 differentially expressed genes, annotated and classified into 50 primary functional categories. Kyoto Encyclopedia of Genes and Genomes analyses revealed 44 differentially expressed genes in nine metabolic pathways. In relation to carbohydrate metabolism pathways, there was downregulation of a polygalacturonase gene, which could reduce cell wall lysis in early pollen germination, and an increase in callose synthase transcripts along with reduced cellulase transcripts. These altered gene expressions responsible for cell wall integrity may inhibit the initiation of pollen tube growth. The onset of tube growth could be further impeded by observed changes in gene expression that potentially influence hormone metabolism (including downregulation of AUXIN RESPONSE FACTOR 18 and EIN3-BINDING F-BOX), reduce mitochondrial function, and alter protein translation. Genes with potential roles in adaptation were also altered in their transcript levels. These included genes encoding the upregulated transcription factor ZmNF-YC2, and the downregulated ZmbHLH13, a negative regulator of jasmonic acid responses. The upregulated flavin enzyme gene DIHYDROLIPOYL DEHYDROGENASE 1, associated with increased levels of reactive oxygen species, is of interest in relating redox homeostasis to stress adaptation. Overall, the analyses identified a suite of genes involved in the development of pollen grains and tubes and responsive to drought stress. The findings enhance understanding of the gene networks underlying compromised pollen viability under drought stress.

EgENOD93 was first identified in a cDNA microarray study of oil palm tissue culture where it was highly expressed in leaf explants with embryogenic potential. Functional characterization via an RNA interference study of its orthologue in Medicago truncatula demonstrated a significant role of this gene in somatic embryo formation. In this study, EgENOD93 was overexpressed in the important model plant Arabidopsis thaliana to investigate the embryogenic potential of EgENOD93 transgenic Arabidopsis explants compared to explants from control plants (pMDC140 and WT). Experiments using leaf explants revealed higher numbers of regenerated shoots at day 27 in all the homozygous transgenic Arabidopsis cultures (Tg01, Tg02 and Tg03) compared to controls. The expression level of EgENOD93 in Arabidopsis cultures was quantified using reverse transcription quantitative real-time PCR (RT-qPCR). The results supported the overexpression of this gene in transgenic Arabidopsis cultures, with 6 and 10 times higher expression of EgENOD93 in callus at Day 9 and Day 20, respectively. Overall, the results support the role of EgENOD93 in the enhancement of shoot regeneration in transgenic Arabidopsis. This together with the previous results observed in oil palm and Medicago truncatula suggests that ENOD93 plays a key role in the induction of somatic embryogenesis. A similarity to early nodulation-like ontogeny is possible.

Plant mitochondria have large genomes to house a small number of key genes. Most mitochondria do not contain a whole genome. Despite these latter characteristics, the mitochondrial genome is faithfully maternally inherited. To maintain the mitochondrial genes—so important for energy production—the fusion and fission of mitochondria are critical. Fission in plants is better understood than fusion, with the dynamin-related proteins (DRP 3A and 3B) driving the constriction of the mitochondrion. How the endoplasmic reticulum and the cytoskeleton are linked to the fission process is not yet fully understood. The fusion mechanism is less well understood, as obvious orthologues are not present. However, there is a recently described gene, MIRO2, that appears to have a significant role, as does the ER and cytoskeleton. Massive mitochondrial fusion (MMF or hyperfusion) plays a significant role in plants. MMF occurs at critical times of the life cycle, prior to flowering, in the enlarging zygote and at germination, mixing the cells’ mitochondrial population—the so-called “discontinuous whole”. MMF in particular aids genome repair, the conservation of critical genes and possibly gives an energy boost to important stages of the life cycle. MMF is also important in plant regeneration, an important component of plant biotechnology.

In Arabidopsis, the actin gene family comprises eight expressed and two non-expressed ACTIN (ACT) genes. Of the eight expressed isoforms, ACT2, ACT7, and ACT8 are differentially expressed in vegetative tissues and may perform specific roles in development. Using tobacco mesophyll protoplasts, we previously demonstrated that actin-dependent clustering of chloroplasts around the nucleus prior to cell division ensures unbiased chloroplast inheritance. Here, we report that actin-dependent chloroplast clustering in Arabidopsis mesophyll protoplasts is defective in act7 mutants, but not act2-1 or act8-2. ACT7 expression was upregulated during protoplast culture whereas ACT2 and ACT8 expression did not substantially change. In act2-1, ACT7 expression increased in response to loss of ACT2, whereas in act7-1, neither ACT2 nor ACT8 expression changed appreciably in response to the absence of ACT7. Semi-quantitative immunoblotting revealed increased actin concentrations during culture, although total actin in act7-1 was only two-thirds that of wild-type or act2-1 after 96 h culture. Over-expression of ACT2 and ACT8 under control of ACT7 regulatory sequences restored normal levels of chloroplast clustering. These results are consistent with a requirement for ACT7 in actin-dependent chloroplast clustering due to reduced levels of actin protein and gene induction in act7 mutants, rather than strong functional specialization of the ACT7 isoform.

Cellular comparisons on three legume species indicate oil body size is factor in mediating oil content. Key transcription factors and oleosins determining seed oil content were identified in somatic embryos of Medicago. Legume seeds provide a significant oil alternative to meat fat needs. Increasing demand for oil nutrition in the context of sustainable crop production has stimulated the exploration of legume seed oil storage regulation. This study investigated the cellular characteristics of seed oil storage using three legume species i.e. Medicago truncatula, Glycine max and Pongamia pinnata representing different oil/protein ratios, and then examined in vitro approaches for assessing strategies in enhancing seed oil storage. A greater range of oil body sizes was in higher oil/protein content species, with highest species having the largest oil bodies; and the smallest oil body size being relatively similar across species, suggesting that the arrangement of oil body size may be factor in mediating oil content. The expression of four key transcription factors i.e. LEC1, L1L, FUS3 and ABI3, and four oleosin genes in determining seed oil content was compared in vivo and in vitro using somatic embryos in Medicago, along with cellular evidence of oil bodies in somatic embryos, indicating that somatic embryos may be suitable models for rapid assessment of seed oil enhancement. This study revealed the cellular characteristics for legume seed oil storage with different nutritional compositions, and identified the associated molecular basis for boosting seed oil storage via regulating oil body size. In addition, somatic embryogenesis may be an effective system for examining oil production by modifying the expression of candidate genes prior to in vivo testing.

KEY MESSAGE:Transcript profiling during the early induction phase of oil palm tissue culture and RNAi studies in a model somatic embryogenesis system showed that EgENOD93 expression is essential for somatic embryogenesis. Micropropagation of oil palm through tissue culture is vital for the generation of superior and uniform elite planting materials. Studies were carried out to identify genes to distinguish between leaf explants with the potential to develop into embryogenic or non-embryogenic callus. Oil palm cDNA microarrays were co-hybridized with cDNA probes of reference tissue, separately with embryo forming (media T527) and non-embryo (media T694) forming leaf explants sampled at Day 7, Day 14 and Day 21. Analysis of the normalized datasets has identified 77, 115 and 127 significantly differentially expressed genes at Day 7, Day 14, and Day 21, respectively. An early nodulin 93 protein gene (ENOD93), was highly expressed at Day 7, Day 14, and Day 21 and in callus (media T527), as assessed by RT-qPCR. Validation of EgENOD93 across tissue culture lines of different genetic background and media composition showed the potential of this gene as an embryogenic marker. In situ RNA hybridization and functional characterization in Medicago truncatula provided additional evidence that ENOD93 is essential for somatic embryogenesis. This study supports the suitability of EgENOD93 as a marker to predict the potential of leaf explants to produce embryogenic callus. Crosstalk among stresses, auxin, and Nod-factor like signalling molecules likely induces the expression of EgENOD93 for embryogenic callus formation.

Chloroplasts (members of the plastid family) and mitochondria are central to the energy cycles of ecosystems and the biosphere. They both contain DNA, organized into nucleoids, coding for critical genes for photosynthetic and respiratory energy production. This review updates the cellular and molecular biology of how chloroplasts, mitochondria, and their genomes in Angiosperms are maintained; particularly in leaf development and maternal inheritance. Maternal inheritance is the common form of transmission to the next generation. Both organelles cannot be derived de novo. Proplastids during very early leaf development develop into chloroplasts with their characteristic thylakoid structure, with the nucleoids associated with the thylakoids. In cell divisions in the leaf primordia and very early leaf development, mitochondria and plastids are duplicated, their nucleoids replicated and segregated, and the population of mitochondria and plastids segregated to daughter cells using the cytoskeleton. To maintain their nucleoids, mitochondria must undergo fusion as well as fission. Chloroplasts are transmitted to the next generation as proplastids where they are maintained in the egg cell but eliminated from the sperm cells. Mitochondria in the apical meristem undergo massive mitochondrial fusion (MMF) prior to floral induction and subsequent maternal inheritance. MMF also occurs again in early germination. MMF encourages DNA repair and recombination, possibly as part of a quality control in each generation. As a further quality control in both chloroplasts and mitochondria, damaged organelles are removed by autophagy. Following consideration of the above, areas requiring further understanding are highlighted.

Grain legumes provide a rich resource of plant nutrition to human diets and are vital for food security and sustainable cropping. Heat stress during flowering has a detrimental effect on legume seed yield, mainly due to irreversible loss of seed number. To start with, we provide an overview of the developmental and physiological basis of controlling seed setting in response to heat stress. It is shown that every single process of seed setting including male and female gametophyte development, fertilization, and early seed/fruit development is sensitive to heat stress, in particular male reproductive development in legume crops is especially susceptible. A series of physiochemical processes including heat shock proteins, antioxidants, metabolites, and hormones centered with sugar starvation are proposed to play a key role in regulating legume seed setting in response to heat stress. The exploration of the molecular mechanisms underlying reproductive heat tolerance is in its infancy. Medicago truncatula, with a small diploid genome, and well-established transformation system and molecular platforms, has become a valuable model for testing gene function that can be applied to advance the physiological and molecular understanding of legume reproductive heat tolerance.

Medicago truncatula is now widely regarded as a legume model where there is an increasing range of genomic resources. Highly regenerable lines have been developed from the wild-type Jemalong cultivar, most likely due to epigenetic changes. These lines with high rates of somatic embryogenesis (SE) can be compared with wild-type where SE is rare. Much of the research has been with the high SE genotype Jemalong 2HA (2HA). SE can be induced from leaf tissue explants or isolated mesophyll protoplasts. In 2HA, the exogenous phytohormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) are central to SE. However, there are interactions with ethylene, abscisic acid (ABA), and gibberellic acid (GA) which produce maximum SE. In the main, somatic embryos are derived from dedifferentiated cells, undergo organellar changes, and produce stem-like cells. There is evidence that the SE is induced as a result of a stress and hormone interaction and this is discussed. In M. truncatula, there are connections between stress and specific up-regulated genes and specific hormones and up-regulated genes during the SE induction phase. Some of the transcription factors have been knocked down using RNAi to show they are critical for SE induction (MtWUSCHEL, MtSERF1). SE research in M. truncatula has utilized high throughput transcriptomic and proteomic studies and the more detailed investigation of some individual genes. In this review, these studies are integrated to suggest a framework and timeline for some of the key events of SE induction in M. truncatula.

Massive mitochondrial fusion (MMF) in germinating arabidopsis seeds, together with earlier studies, suggests a significant role for MMF in the life cycle of flowering plants. MMF is likely to facilitate nucleoid transmission, mitochondrial DNA (mtDNA) recombination, and the homogenization of mitochondrial components, thus providing a type of quality control for mitochondrial populations in new generations.

The ability of plants to regenerate lies in the capacity of differentiated cells to reprogram and re-enter the cell cycle. Reprogramming of cells requires changes in chromatin organisation and gene expression. However, there has been less focus on changes at the post transcription level. We have investigated P-bodies, sites of post transcriptional gene regulation, in plant cell reprogramming in cultured mesophyll protoplasts; by using a YFP-VARICOSE (YFP-VCSc) translational fusion. We showed an early increase in P-body number and volume, followed by a decline, then a subsequent continued increase in P-body number and volume as cell division was initiated and cell proliferation continued. We infer that plant P-bodies have a role to play in reprogramming the mature cell and re-initiating the cell division cycle. The timing of the first phase is consistent with the degredation of messages no longer required, as the cell transits to the division state, and may also be linked to the stress response associated with division induction in cultured cells. The subsequent increase in P-body formation, with partitioning to the daughter cells during the division process, suggests a role in the cell cycle and its re-initiation in daughter cells. P-bodies were shown to be mobile in the cytoplasm and show actin-based motility which facilitates their post-transcriptional role and partitioning to daughter cells.

The proportion of starch, protein and oil in legume seeds is species dependent. The model legume, Medicago truncatula, has predominantly oil and protein stores. To investigate the regulation of seed oil production we compared M. truncatula with M. orbicularis, which has less oil and protein. The types of protein and fatty acids are similar between the two species. Electron microscopy indicated that the size and distribution of the oil bodies in M. orbicularis, is consistent with reduced oil production. M. orbicularis has more extruded endosperm mucilage compared to M. truncatula. The cotyledons have a greater cell wall content, visualized as thicker cell walls. The reduced oil content in M. orbicularis is associated with increased expression of the MtGLABRA2-like (MtGL2) transcription factor, linked to an inverse relationship between mucilage and oil content in Arabidopsis. The expression of the pectin biosynthesis GALACTURONOSYLTRANSFERASE (GAUT) genes, is also increased in M. orbicularis. These increases in extruded mucilage and cell wall storage components in M. orbicularis are accompanied by reduced expression of transcriptional regulators of oil biosynthesis, MtLEAFY COTYLEDON1-LIKE (MtL1L), MtABSCISIC ACID-INSENSITIVE3 (MtABI3), and MtWRINKLED-like (MtWRI), in M. orbicularis. The reduced oil in M. orbicularis, is consistent with increased synthesis of cell wall polysaccharides and decreased expression of master transcription factors regulating oil biosynthesis and embryo maturation. Comparative investigations between these two Medicago species is a useful system to investigate the regulation of oil content and carbon partitioning in legumes.

The seeds of many legume species including soybean, Pongamia pinnata and the model legume Medicago truncatula store considerable oil, apart from protein, in their cotyledons. However, as a group, legume storage strategies are quite variable and provide opportunities for better understanding of carbon partitioning into different storage products. Legumes with their ability to fix nitrogen can also increase the sustainability of agricultural systems. This review integrates the cell biology, biochemistry and molecular biology of oil body biogenesis before considering biotechnology strategies to enhance oil body biosynthesis. Cellular aspects of packaging triacylglycerol (TAG) into oil bodies are emphasized. Enhancing seed oil content has successfully focused on the up-regulation of the TAG biosynthesis pathways using overexpression of enzymes such as diacylglycerol acyltransferase1 and transcription factors such as WRINKLE1 and LEAFY COTYLEDON1. While these strategies are central, decreasing carbon flow into other storage products and maximizing the packaging of oil bodies into the cytoplasm are other strategies that need further examination. Overall there is much potential for integrating carbon partitioning, up-regulation of fatty acid and TAG synthesis and oil body packaging, for enhancing oil levels. In addition to the potential for integrated strategies to improving oil yields, the capacity to modify fatty acid composition and use of oil bodies as platforms for the production of recombinant proteins in seed of transgenic legumes provide other opportunities for legume biotechnology.