Development
Mitochondria, the main energy transducers in plant cells, require the proper assembly of respiratory chain complexes I–V for their function. The NADH dehydrogenase 4 (nad4) gene encodes mitochondrial respiratory chain complex I subunit IV, but the mechanism underlying nad4 transcript splicing is unclear. Here, we report that the P‐type pentatricopeptide repeat (PPR) protein DEFECTIVE KERNEL 43 (DEK43) is responsible for cis‐splicing of the nad4 transcript in maize. We demonstrate that DEK43 localizes to both the nucleus and mitochondria. The mutation of Dek43 resulted in embryo‐lethal and light‐colored defective kernels. Among the 22 mitochondrial group II introns, the splicing efficiency of nad4 introns 1 and 3 was reduced by up to 50% compared to the wild type. The levels of complex I and supercomplex I+III2 were also reduced in dek43. Furthermore, in‐gel NADH dehydrogenase assays indicated that the activities of these complexes were significantly reduced in dek43. Further, the mitochondrial ultrastructure was altered in the mutant. Together, our findings indicate that DEK43, a dual‐localized PPR protein, plays an important role in maintaining mitochondrial function and maize kernel development.
The endoplasmic reticulum (ER) is the major site for protein folding in eukaryotic cells. ER homeostasis is essential for the development of an organism, whereby the unfolded protein response (UPR) within the ER is precisely regulated. ER‐phagy is a newly identified selective autophagic pathway for removal of misfolded or unfolded proteins within the ER in mammalian cells. Sec62, a component of the translocon complex, was recently characterized as an ER‐phagy receptor during the ER stress recovery phase in mammals. In this study, we demonstrated that the Arabidopsis Sec62 (AtSec62) is required for plant development and might function as an ER‐phagy receptor in plants. We showed that AtSec62 is an ER‐localized membrane protein with three transmembrane domains (TMDs) with its C‐terminus facing to the ER lumen. AtSec62 is required for plant development because atsec62 mutants display impaired vegetative growth, abnormal pollen and decreased fertility. atsec62 mutants are sensitive towards tunicamycin (TM)‐induced ER stress, whereas overexpression of AtSec62 subsequently enhances stress tolerance during the ER stress recovery phase. Moreover, YFP‐AtSec62 colocalizes with the autophagosome marker mCh‐Atg8e in ring‐like structures upon ER stress induction. Taken together, these data provide evidence for the pivotal roles of AtSec62 in plant development and ER‐phagy.
Pollen exine contains complex biopolymers of aliphatic lipids and phenolics. Abnormal development of pollen exine often leads to plant sterility. Molecular mechanisms regulating exine formation have been studied extensively but remain ambiguous. Here we report the analyses of three GDSL esterase/lipase protein genes, OsGELP34, OsGELP110, and OsGELP115, for rice exine formation. OsGELP34 was identified by cloning of a male sterile mutant gene. OsGELP34 encodes an endoplasmic reticulum protein and was mainly expressed in anthers during pollen exine formation. osgelp34 mutant displayed abnormal exine and altered expression of a number of key genes required for pollen development. OsGELP110 was previously identified as a gene differentially expressed in meiotic anthers. OsGELP110 was most homologous to OsGELP115, and the two genes showed similar gene expression patterns. Both OsGELP110 and OsGELP115 proteins were localized in peroxisomes. Individual knockout of OsGELP110 and OsGELP115 did not affect the plant fertility, but double knockout of both genes altered the exine structure and rendered the plant male sterile. OsGELP34 is distant from OsGELP110 and OsGELP115 in sequence, and osgelp34 and osgelp110/osgelp115 mutants were different in anther morphology despite both were male sterile. These results suggested that OsGELP34 and OsGELP110/OsGELP115 catalyze different compounds for pollen exine development.
YUC flavin monooxygenases catalyze the rate‐limiting step of auxin biosynthesis. Here we report the vacuolar targeting and degradation of GFP‐YUC1. GFP‐YUC1 fusion expressed in Arabidopsis protoplasts or transgenic plants was primarily localized in vacuoles. Surprisingly, we established that GFP‐YUC1, a soluble protein, was sorted to vacuoles through the ESCRT pathway, which has long been recognized for sorting and targeting integral membrane proteins. We further show that GFP‐YUC1 was ubiquitinated and in this form GFP‐YUC1 was targeted for degradation, a process that was also stimulated by elevated auxin levels. Our findings revealed a molecular mechanism of GFP‐YUC1 degradation and demonstrate that the ESCRT pathway can recognize both soluble and integral membrane proteins as cargoes.
Grain size is a major determinant of cereal grain yields; however, the relevant regulatory mechanisms controlling this trait have not been fully elucidated. The rice (Oryza sativa ) mutant short grain6 (sg6 ) was identified based on its reduced grain length and weight. Here, we functionally characterized the role of SG6 in determining grain size through the regulation of spikelet hull cell division. SG6 encodes a previously uncharacterized plant AT‐rich sequence and zinc‐binding (PLATZ) protein that is ubiquitously localized throughout the cell and is preferentially expressed in the early developing panicles but not in the endosperm. The overexpression of SG6 resulted in significantly larger and heavier grains, as well as increased plant heights, which is consistent with its elevated spikelet hull cell division rate. Yeast two‐hybrid analyses revealed that SG6 interacts with the core cell cycle machinery DP protein and several other putative cell division regulators, consistent with our transcriptomic analysis, which showed that SG6 activates the expression of many DNA replication and cell‐cycle‐related genes. These results confirm the crucial role of SG6 in determining grain size by regulating spikelet hull cell division and provide clues for understanding the functions of PLATZ family proteins and the network regulating cereal grain size.
As sessile organisms, plants perceive, respond, and adapt to the environmental changes for optimal growth and survival. The plant growth and fitness are enhanced by circadian clocks through coordination of numerous biological events. In legume species, nitrogen‐fixing root nodules were developed as the plant organs specialized for symbiotic transfer of nitrogen between microsymbiont and host. Here, we report that the endogenous circadian rhythm in nodules is regulated by MtLHY in legume species Medicago truncatula. Loss of function of MtLHY leads to a reduction in the number of nodules formed, resulting in a diminished ability to assimilate nitrogen. The operation of the 24‐h rhythm in shoot is further influenced by the availability of nitrogen produced by the nodules, leading to the irregulated nyctinastic leaf movement and reduced biomass in mtlhy mutants. These data shed new light on the roles of MtLHY in the orchestration of circadian oscillator in nodules and shoots, which provides a mechanistic link between nodulation, nitrogen assimilation, and clock function.
Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination. Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture. The mutant gene encodes a novel plasma membrane‐localized legume‐lectin receptor kinase that we named OsLecRK‐S.7. OsLecRK‐S.7 was expressed at different levels in all tested tissues and throughout anther development. In vitro kinase assay showed OsLecRK‐S.7 capable of autophosporylation. Mutation in s13283 (E560K) and mutation of the conserved ATP binding site (K418E) both knocked out the kinase activity. Mass spectrometry showed Thr376, Ser378, Thr386, Thr403, and Thr657 to be the autophosphorylation sites. Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees, but did not abolish the gene function in fertility complementation. oslecrk‐s.7 mutant plant overexpressing OsLecRK‐S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers, and these phenotypes were abolished by E560K or K418E mutation. The results indicated that OsLecRK‐S.7 was a key regulator of pollen development.
Water transport from roots to leaves through xylem is important for plant growth and development. Defects in water transport can cause drought stress, even when there is adequate water in the soil. Here, we identified the maize (Zea mays) wilty5 (wi5) mutant, which exhibits marked dwarfing and leaf wilting throughout most of its life cycle under normal growth conditions. wilty5 seedlings exhibited lower xylem conductivity and wilted more rapidly under drought, NaCl, and high temperature treatments than wild‐type plants. Map‐based cloning revealed that WI5 encodes an active endo‐1,4‐β‐xylanase from glycosyl dehydration family 10, which mainly functions in degrading and reorganizing cell wall xylan. Reverse‐transcription polymerase chain reaction and β‐glucuronidase assays revealed that WI5 is highly expressed in stems, especially in internodes undergoing secondary wall assembly. RNA sequencing suggested that WI5 plays a unique role in internode growth. Immunohistochemistry and electron microscopy confirmed that wi5 is defective in xylan deposition and secondary cell wall thickening. Lignin deposition and xylan content were markedly reduced in wi5 compared to the wild‐type plants. Our results suggest that WI5 functions in xylem cell wall thickening through its xylanase activity and thereby regulates xylem water transport, the drought stress response, and plant growth in maize.
Large‐scale production of male sterile seeds can be achieved by introducing a fertility‐restoration gene linked with a pollen‐killer gene into a recessive male sterile mutant. We attempted to construct this system in rice by using a late‐stage pollen‐specific (LSP ) promoter driving the expression of maize α‐amylase gene ZM‐AA1 . To obtain such promoters in rice, we conducted comparative RNA‐seq analysis of mature pollen with meiosis anther, and compared this with the transcriptomic data of various tissues in the Rice Expression Database, resulting in 269 candidate LSP genes. Initial test of nine LSP genes showed that only the most active OsLSP3 promoter could drive ZM‐AA1 to disrupt pollen. We then analyzed an additional 22 LSP genes and found 12 genes stronger than OsLSP3 in late‐stage anthers. The promoters of OsLSP5 and OsLSP6 showing higher expression than OsLSP3 at stages 11 and 12 could drive ZM‐AA1 to inactivate pollen, while the promoter of OsLSP4 showing higher expression at stage 12 only could not drive ZM‐AA1 to disrupt pollen, suggesting that strong promoter activity at stage 11 was critical for pollen inactivation. The strong pollen‐specific promoters identified in this study provided valuable tools for genetic engineering of rice male sterile system for hybrid rice production.
Maize (Zea mays ssp. mays) is a major staple crop, with the highest tonnage among cereal crops worldwide (FAO 2014). Over the past century, maize yields have increased over eight folds in the US central Corn Belt (from 1287 kg ha-1 in the 1930s to 11,084 kg ha-1 in 2017, http://www.fao.org, Duvick 2005b) due to a combination of genetic gain resulting from breeding efforts and improved management practices (such as application of synthetic nitrogen fertilizers, weed and pest control, increased efficiency of harvest equipment, etc.). A major management practice that contributed to the continuous yield increase is continual increases in planting density (from 30,000 plant ha-1 or less in the 1930s to 80,000 plants ha-1 or higher in the 1980s, Duvick 2005a, 2005b).
The polar growth of pollen tubes is essential for the delivery of sperm cells during fertilization in angiosperms. How this polar growth is regulated has been a long‐standing question. An in vitro pharmacological assay previously implicated proton flux in pollen tube growth, although genetic and cellular supporting evidence was lacking. Here, we report that protons form a gradient from the pollen tube tip to the shank region and this gradient is generated by three members of Arabidopsis H+‐ATPases (AHAs). Genetic analysis suggested that these AHAs are essential for pollen tube growth, thus providing new insight into the regulation of polar growth.
In higher plants, lateral organs are usually of determinate growth. It remains largely elusive how the determinate growth is achieved and maintained. Previous reports have shown that Arabidopsis PEAPOD (PPD) proteins suppress proliferation of dispersed meristematic cells partly through a TOPLESS corepressor complex. Here, we identified a new PPD‐interacting partner, LIKE HETEROCHROMATIN PROTEIN1 (LHP1), using the yeast two‐hybrid system, and their interaction is mediated by the chromo shadow domain and the Jas domain in LHP1 and PPD2, respectively. Our genetic data demonstrate that the phenotype of ppd2 lhp1 is more similar to lhp1 than to ppd2 , indicating epistasis of lhp1 to ppd2 . Microarray analysis reveals that PPD2 and LHP1 can regulate expression of a common set of genes directly or indirectly. Consistently, chromatin immunoprecipitation results confirm that PPD2 and LHP1 are coenriched at the promoter region of their targets such as D3‐TYPE CYCLINS and HIGH MOBILITY GROUP A , which are upregulated in ppd2, lhp1 and ppd2 lhp1 mutants, and that PPD s mediate repressive histone 3 lysine‐27 trimethylation at these loci. Taken together, our data provide evidence that PPD and LHP1 form a corepressor complex that regulates lateral organ growth.
The thermosensitive genic male sterile 5 (tms5 ) mutation causes thermosensitive genic male sterility in rice (Oryza sativa ) through loss of RNase ZS1 function, which influences ubiquitin fusion ribosomal protein L40 (Ub L40 ) messenger RNA levels during male development. Here, we used ATAC‐seq, combined with analysis of H3K9ac and H3K4me2, to identify changes in accessible chromatin during fertility conversion of the two‐line hybrid rice Wuxiang S (WXS) derived from a mutant tms5 allele. Furthermore, RNA‐seq and bioinformatic analyses identified specific transcription factors (TFs) in differentially accessible chromatin regions. Among these TFs, only GATA10 targeted Ub L40 . Osgata10 knockout mutations, which resulted in low expression of Ub L40 and a tendency toward male fertility, confirmed that GATA10 regulated fertility conversion via the modulation of Ub L40 . Meanwhile, GATA10 acted as a mediator for interactions with ERF65, which revealed that transcriptional regulation is a complex process involving multiple complexes of TFs, namely TF modules. It appears that the ERF141/MADS7/MADS50/MYB modules affect metabolic processes that control anther and pollen development, especially cell wall formation. Our analysis revealed that these modules directly or indirectly affect metabolic pathway‐related genes to coordinate plant growth with proper anther development, and furthermore, that GATA10 regulates fertility conversion via the modulation of Ub L40 expression.
Self‐incompatibility (SI) is a genetic mechanism that rejects self‐pollen and thus prevents inbreeding in some hermaphroditic angiosperms. In the Brassicaceae, SI involves a pollen‐stigma recognition system controlled by a single locus known as the S locus, which consists of two highly polymorphic genes that encode S‐locus cysteine‐rich protein (SCR) and S‐receptor kinase (SRK). When self‐pollen lands on the stigma, the S‐haplotype‐specific interaction between SCR and SRK triggers SI. Here, we show that the GATA transcription factor BnA5.ZML1 suppresses SI responses in Brassica napus and is induced after compatible pollination. The loss‐of‐function mutant bna5.zml1 displays reduced self‐compatibility. In contrast, overexpression of BnA5.ZML1 in self‐incompatible stigmas leads to a partial breakdown of SI responses, suggesting that BnA5.ZML1 is a stigmatic compatibility factor. Furthermore, the expression levels of SRK and ARC1 are up‐regulated in bna5.zml1 mutants, and they are down‐regulated in BnA5.ZML1 overexpressing lines. SRK affects the cellular localization of BnA5.ZML1 through direct protein–protein interaction. Overall, our findings highlight the fundamental role of BnA5.ZML1 in SI responses in B. napus , establishing a direct interaction between BnA5.ZML1 and SRK in this process.
Imitation Switch (ISWI) chromatin remodelers are known to function in diverse multi‐subunit complexes in yeast and animals. However, the constitution and function of ISWI complexes in Arabidopsis thaliana remain unclear. In this study, we identified forkhead‐associated domain 2 (FHA2) as a plant‐specific subunit of an ISWI chromatin‐remodeling complex in Arabidopsis. By in vivo and in vitro analyses, we demonstrated that FHA2 directly binds to RLT1 and RLT2, two redundant subunits of the ISWI complex in Arabidopsis. The stamen filament is shorter in the fha2 and rlt1/2 mutants than in the wild type, whereas their pistil lengths are comparable. The shorter filament, which is due to reduced cell size, results in insufficient pollination and reduced fertility. The rlt1/2 mutant shows an early‐flowering phenotype, whereas the phenotype is not shared by the fha2 mutant. Consistent with the functional specificity of FHA2, our RNA‐seq analysis indicated that the fha2 mutant affects a subset of RLT1/2‐regulated genes that does not include genes involved in the regulation of flowering time. This study demonstrates that FHA2 functions as a previously uncharacterized subunit of the Arabidopsis ISWI complex and is exclusively involved in regulating stamen development and plant fertility.
