To determine the proteomic-level responses of drought tolerant wild wheat (Triticum boeoticum), physiological and comparative proteomic analyses were conducted using the roots and the leaves of control and short term drought-stressed plants. Drought stress was imposed by transferring hydroponically grown seedlings at the 3-leaf stage into 1/2 Hoagland solution containing 20% PEG-6000 for 48 h. Root and leaf samples were separately collected at 0 (control), 24, and 48 h of drought treatment for analysis. Physiological analysis indicated that abscisic acid (ABA) level was greatly increased in the drought-treated plants, but the increase was greater and more rapid in the leaves than in the roots. The net photosynthetic rate of the wild wheat leaves was significantly decreased under short-term drought stress. The deleterious effects of drought on the studied traits mainly targeted photosynthesis. Comparative proteomic analysis identified 98 and 85 differently changed protein spots (DEPs) (corresponding to 87 and 80 unique proteins, respectively) in the leaves and the roots, respectively, with only 6 mutual unique proteins in the both organs. An impressive 86% of the DEPs were implicated in detoxification and defense, carbon metabolism, amino acid and nitrogen metabolism, proteins metabolism, chaperones, transcription and translation, photosynthesis, nucleotide metabolism, and signal transduction. Further analysis revealed some mutual and tissue-specific responses to short-term drought in the leaves and the roots. The differences of drought-response between the roots and the leaves mainly included that signal sensing and transduction-associated proteins were greatly up-regulated in the roots. Photosynthesis and carbon fixation ability were decreased in the leaves. Glycolysis was down-regulated but PPP pathway enhanced in the roots, resulting in occurrence of complex changes in energy metabolism and establishment of a new homeostasis. Protein metabolism was down-regulated in the roots, but enhanced in the leaves. These results will contribute to the existing knowledge on the complexity of root and leaf protein changes that occur in response to drought, and also provide a framework for further functional studies on the identified proteins.

Background: The Arabidopsis var2 mutant displays a unique green and white/yellow leaf variegation phenotype and lacks VAR2, a chloroplast FtsH metalloprotease. We are characterizing second-site var2 genetic suppressors as means to better understand VAR2 function and to study the regulation of chloroplast biogenesis. Results: In this report, we show that the suppression of var2 variegation in suppressor line TAG-11 is due to the disruption of the SUPPRESSOR OF VARIEGATION3 (SVR3) gene, encoding a putative TypA-like translation elongation factor. SVR3 is targeted to the chloroplast and svr3 single mutants have uniformly pale green leaves at 22°C. Consistent with this phenotype, most chloroplast proteins and rRNA species in svr3 have close to normal accumulation profiles, with the notable exception of the Photosystem II reaction center D1 protein, which is present at greatly reduced levels. When svr3 is challenged with chilling temperature (8°C), it develops a pronounced chlorosis that is accompanied by abnormal chloroplast rRNA processing and chloroplast protein accumulation. Double mutant analysis indicates a possible synergistic interaction between svr3 and svr7, which is defective in a chloroplast pentatricopeptide repeat (PPR) protein. Conclusions: Our findings, on one hand, reinforce the strong genetic link between VAR2 and chloroplast translation, and on the other hand, point to a critical role of SVR3, and possibly some aspects of chloroplast translation, in the response of plants to chilling stress.

The growth of higher plants is under complex regulation to ensure the elaboration of developmental programmes under a changing environment. To dissect these regulatory circuits, we carried out genetic screens for Arabidopsis abnormal shoot (abs) mutants with altered shoot development. Here, we report the isolation of two dominant mutants, abs3-1D and abs4-1D, through activation tagging. Both mutants showed a ‘bushy’ loss of apical dominance phenotype. ABS3 and ABS4 code for two closely related putative Multidrug and Toxic Compound Extrusion (MATE) family of efflux transporters, respectively. ABS3 and ABS4, as well as two related MATE genes, ABS3-Like1 (ABS3L1) and ABS3L2, showed diverse tissue expression profiles but their gene products all localized to the late endosome/prevacuole (LE/PVC) compartment. The over-expression of these four genes individually led to the inhibition of hypocotyl cell elongation in the light. On the other hand, the quadruple knockout mutant (mateq) showed the opposite phenotype of an enhanced hypocotyl cell elongation in the light. Hypocotyl cell elongation and de-etiolation processes in the dark were also affected by the mutations of these genes. Exogenously applied sucrose attenuated the inhibition of hypocotyl elongation caused by abs3-1D and abs4-1D in the dark, and enhanced the hypocotyl elongation of mateq under prolonged dark treatment. We determined that ABS3 genetically interacts with the photoreceptor gene PHYTOCHROME B (PHYB). Our results demonstrate that ABS3 and related MATE family transporters are potential negative regulators of hypocotyl cell elongation and support a functional link between the endomembrane system, particularly the LE/PVC, and the regulation of plant cell elongation.

The development of functional chloroplasts relies on the fine coordination of expressions of both nuclear and chloroplast genomes. We have been using the Arabidopsis yellow variegated (var2) leaf variegation mutant as a tool to dissect the regulation of chloroplast development. In this work, we screened for var2 genetic enhancer modifiers termed enhancer of variegation (evr) mutants and report the characterization of the first EVR locus, EVR1. We showed that EVR1 encodes the cytosolic 80S ribosome 40S small subunit protein RPS21B and the loss of EVR1 causes the enhancement of var2 leaf variegation. We further demonstrated that combined S21 activities from EVR1 and its close homolog, EVR1L1, are essential for Arabidopsis, and they act redundantly in regulating leaf development and var2 leaf variegation. Moreover, using additional cytosolic ribosomal protein mutants, we showed that although mutations in cytosolic ribosomal proteins all enhance var2 leaf variegation to varying degrees, 40S subunit appears to have a more profound role over 60S subunit in regulating VAR2-mediated chloroplast development. Comprehensive genetic analyses with var2 suppressors that are defective in chloroplast translation established that the enhancement of var2 leaf variegation by cytosolic ribosomal protein mutants is dependent on chloroplast translation. Based on our data, we propose a model that incorporates the suppression and enhancement of var2 leaf variegation, and hypothesize that VAR2/AtFtsH2 may be intimately involved in the balancing of cytosolic and chloroplast translation programs during chloroplast biogenesis.

Chloroplast development requires the coordinated expressions of nuclear and chloroplast genomes, and both anterograde and retrograde signals exist and work together to facilitate this coordination. We have utilized the Arabidopsis yellow variegated (var2) mutant as a tool to dissect the genetic regulatory network of chloroplast development. Here, we report the isolation of a new (to our knowledge) var2 genetic suppressor locus, SUPPRESSOR OF VARIEGATION9 (SVR9). SVR9 encodes a chloroplast-localized prokaryotic type translation initiation factor 3 (IF3). svr9-1 mutant can be fully rescued by the Escherichia coli IF3 infC, suggesting that SVR9 functions as a bona fide IF3 in the chloroplast. Genetic and molecular evidence indicate that SVR9 and its close homolog SVR9-LIKE1 (SVR9L1) are functionally interchangeable and their combined activities are essential for chloroplast development and plant survival. Interestingly, we found that SVR9 and SVR9L1 are also involved in normal leaf development. Abnormalities in leaf anatomy, cotyledon venation patterns, and leaf margin development were identified in svr9-1 and mutants that are homozygous for svr9-1 and heterozygous for svr9l1-1 (svr9-1 svr9l1-1/+). Meanwhile, as indicated by the auxin response reporter DR5:GUS, auxin homeostasis was disturbed in svr9-1, svr9-1 svr9l1-1/+, and plants treated with inhibitors of chloroplast translation. Genetic analysis established that SVR9/SVR9L1-mediated leaf margin development is dependent on CUP-SHAPED COTYLEDON2 activities and is independent of their roles in chloroplast development. Together, our findings provide direct evidence that chloroplast IF3s are essential for chloroplast development and can also regulate leaf development.